Delivery of therapeutic compositions using ultrasound

ABSTRACT

Therapeutic compositions are delivered to a target site using a catheter which includes at least one ultrasound transducer coupled to an energy source. The therapeutic compositions include genetic material and the target site may be a DNA with affinity for the genetic material.

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. application Ser. No.09/620,701, filed Jul. 20, 2000 which is a Divisional of U.S.application Ser. No. 09/158,316, filed Sep. 21, 1998, now U.S. Pat. No.6,176,842 which is a Continuation-in-Part of U.S. application Ser. No.09/129,980; filed Aug. 5, 1998, and a Continuation-In-Part of Japaneseapplication number HO9-255814, filed Sep. 19, 1997, entitled DrugCarrier and Method of Using Same and a Continuation-In-Part of U.S.application Ser. No. 08/972,846, filed Nov. 18, 1997, now abandoned,which is a Continuation of U.S. application Ser. No. 08/611,105, filedMar. 5, 1996, now abandoned, which claims priority to Japaneseapplication number P07-048710; filed Mar. 8, 1995. Each of the aboveapplications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method that utilizesultrasonic vibration to perform various types of therapies, andparticularly to a therapy-accelerating substance that controls theadministration of a drug by utilizing ultrasonic vibration at specificlocations within the body to perform the release of the drugeffectively. The drug may include a nucleic acid material such as a DNA,RNA, or one or more oligonucleotides.

[0004] 2. Description of the Related Art

[0005] Current methods of treating cancer can be broadly divided intotwo methods: the removal of the cancerous tissue by surgery or thekilling of the cancerous cells with anti-cancer drugs. The removal ofcancerous tissue can only be performed in cases in which the canceroccurs in limited small areas and no metastasis is found. On the otherhand, cancer chemotherapy has extremely strong side effects such asnausea, impairment of kidney function and impairment of liver functionwhich happens often with the administration of large doses of drugs.Moreover, cancer reacts only to drugs in high concentrations, so cancerchemotherapy has not demonstrated very good clinical results.

[0006] Many different approaches to solving this problem are currentlybeing attempted. The method called “missile therapy” (a term used byJapanese physicians to refer to types of cancer therapy where a payloadof drug or radioactive isotope is delivered accurately to the site of acancer in the manner of a military “smart bomb” or missile) usinganti-cancer drugs is a method that uses anti-cancer drugs bound toantibodies that selectively bind with the cancerous cells, so that theanti-cancer drugs act in a concentrated manner on the cancerous cells,but this method has not yet achieved adequate results.

[0007] On the other hand, a method has been proposed in whichanti-cancer drugs enclosed in capsules or other drug carriers made ofspecific materials are injected into the body and the shells are made torupture within blood vessels near the cancer so that a highconcentration of anti-cancer drugs are administered to limited areas.Its effectiveness has been demonstrated experimentally.

[0008] However, methods of efficiently rupturing the capsule shells usedin this method have not been established. Up until now, research hasbeen conducted on embedding polymer-based temperature sensors, pHsensors and the like in the capsule shells so that the release of drugsis induced under certain temperature or pH conditions, but arbitrarilysetting the temperature and pH conditions near the location of the tumoris extremely difficult.

[0009] In addition, methods have been proposed by which the drugs arereleased from the capsules when the capsule shells are ruptured by shockwaves or ultrasonic energy applied from outside. For example, thespecification of U.S. Pat. No. 5,580,575 recites a method whereinliposomes containing a gas and drugs are ruptured by ultrasound at aspecific location in the body of the patient. However, rupturing aliposome or other capsule mechanically with the vibration of ultrasoundalone in this manner requires irradiation with high-intensityultrasound, and the resonance frequency is determined depending on theamount of gas within the capsule, so it is difficult to rupture thecapsule with ultrasound using frequencies other than the resonancefrequency.

[0010] In this manner, the use of acoustical energy requiresconsiderable precision in the setting of the irradiation conditions, soit has not been easy to achieve the precise ultrasound frequency andintensity required in the location of the tumor.

[0011] The problem addressed by the present invention is to provide adrug carrier and method of using same that is able to achieve thedelivery of high concentrations of anti-cancer drugs or other drugs tospecific locations by means of capsules or other drug carriers carryingsaid anti-cancer drugs or other drugs that are reliably and simplyruptured using ultrasound at specific locations such as in blood vesselswithin cancerous tissue or on the surface of the skin. In order to solvethe aforementioned problem, a drug carrier contains anultrasound-sensitive substance.

[0012] It is frequently desirable to kill targeted biological tissuessuch as tumors and atheroma. One technique for causing targeted tissuedeath is called photodynamic therapy which requires the use of lightactivated drugs. Light activated drugs are inactive until exposed tolight of particular wavelengths, however, upon exposure to light of theappropriate wavelength, light activated drugs can exhibit a cytotoxiceffect on the tissues where they are localized. It has been postulatedthat the cytotoxic effect is a result of the formation of singlet oxygenon exposure to light.

[0013] Photodynamic therapy begins with the systemic administration of aselected light activated drug to a patient. At first, the drug dispersesthroughout the body and is taken up by most tissues within the body.After a period of time usually between 3 and 48 hours, the drug clearsfrom most normal tissue and is retained to a greater degree in lipidrich regions such as the liver, kidney, tumor and atheroma. A lightsource, such as a fiber optic, is then directed to a targeted tissuesite which includes the light activated drug. The tissues of the tissuesite are then exposed to light from the light source in order toactivate any light activated drugs within the tissue site. Theactivation of the light activated drug causes tissue death within thetissue site.

[0014] Several difficulties can be encountered during photodynamictherapy. For instance, since the light activated drug is typicallyadministered systemically, the concentration of the light activated drugwithin the targeted tissue site is limited by the quantity of lightactivated drug administered. The concentration of the light activateddrug within a tissue site can also be limited by the degree of selectiveuptake of the light activated drug into the tissue site. Specifically,if the targeted tissue site does not selectively uptake the lightactivated drug, the concentration of light activated drug within thetissue site can be too low for an effective treatment.

[0015] An additional problem associated with photodynamic therapyconcerns depth of treatment. Light cannot penetrate deeply into opaquetissues. As a result, the depth that light penetrates most tissue sitesis limited. This limited depth can prevent photodynamic therapy frombeing used to treat tissues which are located deeply in the interior ofa tissue site.

[0016] There is currently a need for a method and apparatus which can beused to cause death to tissues death deep within a tissue site. When themethod and apparatus employ light activated drugs, the method andapparatus should be able to provide an appropriate concentration oflight activated drug within the tissue site.

SUMMARY OF THE INVENTION

[0017] An object for an embodiment of the invention is causing tissuedeath within a tissue site.

[0018] Another object for an embodiment of the present invention islocally delivering a light activated drug to a tissue site andactivating the light activated drug.

[0019] Yet another object for an embodiment of the present invention islocally delivering a light activated drug to a tissue site anddelivering ultrasound energy to the delivered light activated drug toactivate the light activated drug.

[0020] A further object for an embodiment of the present invention isusing a catheter to locally deliver a light activated drug to a tissuesite and delivering ultrasound energy from an ultrasound element on thecatheter to activate the light activated drug.

[0021] Yet a further object for an embodiment of the present inventionis including the light activated drug in an emulsion, locally deliveringthe emulsion to a tissue site and delivering ultrasound energy to thelight activated drug within the tissue site to activate the lightactivated drug.

[0022] Even a further object for an embodiment of the present inventionis including the light activated drug in a liposome, locally deliveringthe liposome to a tissue site and delivering ultrasound energy to thelight activated drug within the tissue site to activate the lightactivated drug.

[0023] An additional object for an embodiment of the present inventionis including the light activated drug in an aqueous solution, locallydelivering the aqueous solution to a tissue site and deliveringultrasound energy to the light activated drug within the tissue site toactivate the light activated drug.

[0024] Yet a further object for an embodiment of the present inventionis including the light activated drug in an emulsion, systemicallydelivering the emulsion, providing the light activated drug sufficienttime to localize within a tissue site and delivering ultrasound energyto the light activated drug within the tissue site to activate the lightactivated drug.

[0025] Even a further object for an embodiment of the present inventionis including the light activated drug in liposomes, systemicallydelivering the liposomes, providing the light activated drug sufficienttime to localize within a tissue site and delivering ultrasound energyto the light activated drug within the tissue site to activate the lightactivated drug.

[0026] An additional object for an embodiment of the present inventionis including the light activated drug in an aqueous solution,systemically delivering the aqueous solution, providing the lightactivated drug sufficient time to localize within a tissue site anddelivering ultrasound energy to the light activated drug within thetissue site to activate the light activated drug.

[0027] Another object for an embodiment of the present invention iscoupling a site directing molecule to a light activated drug, locallydelivering the light activated drug to a tissue site and activating thelight activated drug within the tissue site.

[0028] Yet another object for an embodiment of the invention isproviding a catheter for locally delivering a media including a lightactivated drug to a tissue site. The catheter including an ultrasoundassembly configured to activate the light activated drug within thetissue site.

[0029] A further object for an embodiment of the invention is providinga catheter for delivering a media including a light activated drug to atissue site. The catheter including an ultrasound assembly for reducingexposure of the light activated drug to ultrasound energy until thelight activated drug has been delivered from within the catheter.

[0030] A kit for causing tissue death within a tissue site is disclosed.The kit includes a media with a light activated drug activatable uponexposure to a particular level of ultrasound energy. The kit alsoincludes a catheter with a lumen coupled with a media delivery portthrough which the light activated drug can be locally delivered to thetissue site. The ultrasound transducer is configured to transmit thelevel of ultrasound energy which activates the light activated drug withsufficient power that the ultrasound energy can penetrate the tissuesite.

[0031] A method for causing tissue death in a subdermal tissue site isalso disclosed. The method includes providing a catheter for locallydelivering a light activated drug to the subdermal tissue site, thecatheter including an ultrasound transducer. The method also includeslocally delivering the light activated drug to the tissue site;producing ultrasound energy from the ultrasound transducer; anddirecting the ultrasound energy to the subdermal tissue site followingpenetration of the light activated drug into the subdermal tissue siteto activate at least a portion of the light activated drug within thesubdermal tissue site.

[0032] A method for activating a light activated drug is also disclosed.The method includes providing a catheter with an ultrasound transducer.The method also includes introducing the light activated drug into apatient's body where a subdermal tissue site absorbs at least a portionof the light activated drug; producing ultrasound energy; directing theultrasound energy to the light activated containing subdermal tissuesite including the light activated drug; and activating at least aportion of the light activated drug in the subdermal selected tissuesite.

[0033] A method for releasing a therapeutic from a microbubble is alsodisclosed. The method includes providing a microbubble with a lightactivated drug activatable upon exposure to ultrasound energy; anddelivering ultrasound energy to the microbubble at a frequency andintensity which activates the light activated drug to cause a rupture ofthe microbubble.

[0034] A microbubble is also disclosed. The microbubble includes asubstrate defining a shell of the microbubble and having a thicknesspermitting hydraulic transport of the microbubble. The microbubble alsoincludes a light activated drug activatable upon exposure to ultrasoundenergy. Activation of the light activated drug causes a disruption inthe shell sufficient to cause a rupture of the microbubble. Themicrobubble further includes a therapeutic releasable from themicrobubble upon rupture of the microbubble and yielding a therapeuticeffect upon release from the microbubble.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1A is a side view of a catheter for locally delivering amedia including a light activated drug to a tissue site.

[0036]FIG. 1B is an axial cross section of an ultrasound assembly foruse with the catheter shown in FIG. 1A.

[0037]FIG. 1C is a lateral cross section of an ultrasound assembly foruse with the catheter shown in FIG. 1A.

[0038]FIG. 2A is a side view of a catheter having an elongated body andan ultrasound assembly which is flush with the elongated body.

[0039]FIG. 2B is an axial cross section of the ultrasound assemblyillustrated in FIG. 2A.

[0040]FIG. 2C is a lateral cross section of the ultrasound assemblyillustrated in FIG. 2A.

[0041]FIG. 3A illustrates a catheter with a utility lumen and a secondutility lumen.

[0042]FIG. 3B is an axial cross section of the ultrasound assemblyillustrated in the catheter of FIG. 3A.

[0043]FIG. 4A is a side view of a catheter including a plurality ofultrasound assemblies.

[0044]FIG. 4B is a cross section of an ultrasound assembly included on acatheter with a plurality of utility lumens.

[0045]FIG. 4C is a cross section of an ultrasound assembly included on acatheter with a plurality of utility lumens.

[0046]FIG. 5A is a side view of a catheter including a balloon.

[0047]FIG. 5B is a cross section of a catheter with a balloon whichinclude an ultrasound assembly.

[0048]FIG. 6A is a side view of a catheter with a balloon positioneddistally relative to an ultrasound assembly.

[0049]FIG. 6B is a side view of a catheter with an ultrasound assemblypositioned distally relative to a balloon.

[0050]FIG. 6C is a cross section of a catheter with an ultrasoundassembly positioned at the distal end of the catheter.

[0051]FIG. 7A is a side view of a catheter with a media delivery portpositioned between an ultrasound assembly and a balloon.

[0052]FIG. 7B is a side view of a catheter with an ultrasound assemblypositioned between a media delivery port and a balloon.

[0053]FIG. 7C is a cross section of a catheter with an ultrasoundassembly positioned at the distal end of the catheter.

[0054]FIG. 8A is a side view of a catheter including a media deliveryport and an ultrasound assembly positioned between first and secondballoons.

[0055]FIG. 8B is a side view of a catheter including a media deliveryport and an ultrasound assembly positioned between first and secondballoons.

[0056]FIG. 8C is a cross section of a balloon included on a catheterhaving a first and second balloon.

[0057]FIG. 9A illustrates an ultrasound assembly positioned adjacent toa tissue site and microbubbles delivered via a utility lumen.

[0058]FIG. 9B illustrates an ultrasound assembly positioned adjacent toa tissue site and a media delivered via a media delivery port.

[0059]FIG. 9C illustrates an ultrasound assembly positioned adjacent toa tissue site and a media delivered via a media delivery port while aguidewire is positioned in a utility lumen.

[0060]FIG. 9D illustrates a catheter including a balloon positionedadjacent to a tissue site.

[0061]FIG. 9E illustrates a catheter including a balloon expanded intocontact with a tissue site.

[0062]FIG. 9F illustrates a catheter with an ultrasound assembly outsidea balloon positioned at a tissue site.

[0063]FIG. 9G illustrates the balloon of FIG. 9F expanded into contactwith a vessel so as to occlude the vessel.

[0064]FIG. 9H illustrates a catheter with an ultrasound assembly outsidea first and second balloon positioned at a tissue site.

[0065]FIG. 9I illustrates the first and second balloon of FIG. 9Hexpanded into contact with a vessel so as to occlude the vessel.

[0066]FIG. 10A is a cross section of an ultrasound assembly according tothe present invention.

[0067]FIG. 10B is a cross section of an ultrasound assembly according tothe present invention.

[0068]FIG. 10C illustrates a support member with integral supports.

[0069]FIG. 10D illustrates a support member which is supported by anouter coating.

[0070]FIG. 1A is a cross section of an ultrasound assembly including twoconcentric ultrasound transducers in contact with one another.

[0071]FIG. 11B is a cross section of an ultrasound assembly includingtwo separated and concentric ultrasound transducers.

[0072]FIG. 11C is a cross section of an ultrasound assembly includingtwo ultrasound transducers where a chamber is defined between one of theultrasound transducers and an elongated body.

[0073]FIG. 11D is a cross section of an ultrasound assembly includingtwo longitudinally adjacent ultrasound transducers in physical contactwith one another.

[0074]FIG. 11E is a cross section of an ultrasound assembly includingtwo separated and longitudinally adjacent ultrasound transducers.

[0075]FIG. 11F is a cross section of an ultrasound assembly includingtwo longitudinally adjacent ultrasound transducers with a single chamberpositioned between both ultrasound transducers and an elongated body.

[0076]FIG. 11G is a cross section of an ultrasound assembly includingtwo longitudinally adjacent ultrasound transducers with differentchambers positioned between each ultrasound transducers and an elongatedbody.

[0077]FIG. 11H is a cross section of an ultrasound assembly includingtwo longitudinally adjacent ultrasound transducers in contact with oneanother and having a single chamber positioned between each ultrasoundtransducers and an elongated body.

[0078]FIG. 12A is a cross section of a catheter which includes anultrasound assembly module which is independent of a first cathetercomponent and a second catheter component.

[0079]FIG. 12B illustrates the first and second catheter componentscoupled with the ultrasound assembly module.

[0080]FIG. 12C is a cross section of an ultrasound assembly which isintegral with a catheter.

[0081]FIG. 13A is a cross section of an ultrasound assembly configuredto radiate ultrasound energy in a radial direction. The lines whichdrive the ultrasound transducer pass through a utility lumen in thecatheter.

[0082]FIG. 13B is a cross section of an ultrasound assembly configuredto radiate ultrasound energy in a radial direction. The lines whichdrive the ultrasound transducer pass through line lumens in thecatheter.

[0083]FIG. 13C is a cross section of an ultrasound assembly configuredto longitudinally radiate ultrasound energy. The distal portion of oneline travels proximally through the outer coating.

[0084]FIG. 13D is a cross section of an ultrasound assembly configuredto longitudinally transmit ultrasound energy. The distal portion of oneline travels proximally through a line lumen in the catheter.

[0085]FIG. 14A illustrates ultrasound transducers connected in parallel.

[0086]FIG. 14B illustrates ultrasound transducers connected in series.

[0087]FIG. 14C illustrates ultrasound transducers connected with acommon line.

[0088]FIG. 15 illustrates a circuit for electrically couplingtemperature sensors.

[0089]FIG. 16 illustrates a feedback control system for use with acatheter including an ultrasound assembly.

[0090] FIGS. 17A-N illustrate pyrrole-based macrocyclic classes of lightemitting drugs.

[0091]FIG. 17B-2 illustrates possible texaphyrin derivation sites.

[0092] FIGS. 18A-F illustrate the formula of preferred light emittingdrugs for use with media including microbubbles.

[0093]FIG. 19 illustrates a formula for a porphyrin group.

[0094] FIGS. 20A-D illustrate the formula of four preferred forms of thehydro-monobenzoporphyrin derivatives of the green porphyrins illustratedin formulae 3 and 4 of FIG. 18.

[0095] FIGS. 21A-B illustrate the formulae for specific examples ofpyrrole-based macrocycle derivatives and xanthene derivatives which arepreferred for inclusion in microbubbles to enhance rupture of themicrobubbles upon activation.

[0096]FIG. 22 shows a sectional structural drawing of one embodiment ofa drug carrier.

[0097]FIG. 23 shows a sectional structural drawing of another embodimentof a drug carrier.

[0098]FIG. 24 shows a sectional structural drawing of still anotherembodiment of a drug carrier.

[0099]FIG. 25 shows a sectional drawing showing the mounting of theultrasound generator used in the preferred embodiment of the presentinvention.

[0100]FIG. 26 shows a sectional drawing showing one embodiment of atherapeutic ultrasound generator.

[0101]FIG. 27 shows an enlarged sectional drawing used to explain thecase in which the drug carriers of the present invention are applied tothrombolytic therapy.

[0102]FIG. 28 shows an enlarged sectional drawing used to explain thecase in which the drug carriers of the present invention are applied toblood vessel therapy.

[0103]FIG. 29 shows a sectional drawing of a transdermal administrationapparatus that applies the drug carriers of the present invention.

[0104] FIGS. 30A-I schematically summarize the synthesis of anoligonucleotide conjugate of a texaphyrin metal complex.

[0105] FIGS. 31A-H illustrate the covalent coupling of texaphyrin metalcomplexes with amine, thiol, or hydroxy linked oligonucleotides.

[0106] FIGS. 32A-F illustrate the synthesis of diformyl monoic acid andoligonucleotide conjugate.

[0107] FIGS. 33A-J illustrate the synthesis of a texaphyrin based lightactivated drug.

[0108]FIG. 34 illustrates the formula for tin ethyl etiopurpurin(SnEt₂).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0109] The present invention relates to a method and catheter fordelivering a light activated drug to a tissue site and deliveringultrasound energy to the light activated drug within the tissue site.Since many light activated drugs are also activated by ultrasoundenergy, the delivery of ultrasound energy to the light activated drugactivates the light activated drug within the tissue site. Similar toactivation of a light activated drug by light, activation by ultrasoundcauses death of tissues within the tissue site. The tissue death isbelieved to result from the release of a singlet oxygen. Suitable tissuesites include, but are not limited to, atheroma, cancerous tumors,thrombi and potential restenosis sites. A potential restenosis site is atissue site where restenosis is likely to occur such as the portion ofvessels previously treated by balloon angioplasty. In contrast to light,ultrasound energy can be transmitted through opaque tissues. As aresult, the ultrasound energy can be used to treat tissues which aredeeper within a tissue site than could be treated via light activation.

[0110] One explanation for the activation of light activated drugs viathe application of ultrasound is a result of cavitation. Cavitation isknown to occur when ultrasonic energy above a certain threshold isapplied to a liquid. The mechanism of generation of cavitation isdescribed in Apfel, Robert E., “Sonic Effervescence: Tutorial onAcoustic Cavitation” Journal of Acoustic Society of America 101 (3):1227-1237 (March 1997) and Atchley A., Crum L., “Ultrasound—ItsChemical, Physical and Biological Effects: Acoustic Cavitation andBubble Dynamics,” pp. 1-64, 1988 VCH Publishers, New York (1998).

[0111] Cavitation results when gas dissolved in a solution forms bubblesunder certain types of acoustic vibration. Cavitation can also occurwhen small bubbles already present in the solution oscillate orrepeatedly enlarge and contract to become bubbles. When the size ofthese cavitation bubbles reaches a size that cannot be maintained, theysuddenly collapse and release various types of energy. The various typesof energy include, but are not limited to, mechanical energy, visiblelight, ultraviolet light and other types of electromagnetic radiation.Heat, plasma, magnetic fields, shock waves, free radicals, heat andother forms of energy are also thought to be generated locally. Thelight activated drug is believed to be activated by at least one of thevarious forms of energy generated at the time of cavitation collapse.

[0112] The delivery of light activated drug to the tissue site can bethrough traditional systemic administration of a media including thelight activated drug or can be performed through localized delivery ofthe media. Localized delivery can be achieved through injection into thetissue site or through other traditional localized delivery techniques.A preferred delivery technique is using a catheter which includes amedia delivery lumen coupled with a media delivery port. The cathetercan be positioned such that the media delivery port is within the tissuesite or is adjacent to the tissue site via traditionalover-the-guidewire techniques. The media can then be locally deliveredto the tissue site through the media delivery port.

[0113] The localized delivery of the light activated drug to the tissuesite serves to localize the light activated drug within the tissue siteand can reduce the amount of light activated drug which concentrates intissues outside the tissue site. Further, localized delivery of thelight activated drug can serve to increase the concentration of thelight activated drug within the tissue site above levels which would beachieved through systemic delivery of the light activated drug.Alternatively, the same concentration of light activated drug within thetissue site as would occur through systemic administration can beachieved by introducing smaller amounts of light activated drug into apatient's body.

[0114] Localized delivery of the light activated drug also permitstreatment of tissue sites which do not have selective uptake of thelight activated drug. As discussed above, many light activated drugs,such as the texaphyrins, are taken up by most tissues within the bodyand later localize within lipid rich tissues. As a result, a non-lipidrich tissue site can be treated by delivering the ultrasound energy tothe tissue site before the light activated drug has an opportunity tolocalize in lipid rich tissues.

[0115] Localized delivery is also advantageous when the tissue site islipid rich such as in an atheroma or a tumor. The localized delivery ofthe light activated drug combined with the inherent affinity of thelight activated drug for tissue site can result in a high degree oflocalization of the light activated drug within lipid rich tissue sites.

[0116] To increase localization of the light activated drug within thetissue site, the light activated drug can be coupled with a sightdirecting molecule to form a light activated drug conjugate. The sitedirecting molecule is chosen so the light activated drug conjugatespecifically binds with the tissue site when the light activated drugconjugate is contacted with the tissue site under physiologicalconditions of temperature and pH. The specific binding may result fromspecific electrostatic, hydrophobic, entropic, or other interactionsbetween certain residues on the conjugate and specific residues on thetissue site.

[0117] In one preferred embodiment, the light activated drug includes anoligonucleotide acting as a site specific molecule coupled with atexaphyrin. The oligonucleotide can have an affinity for a targeted siteon a DNA strand. For instance, the oligonucleotide can be designed tohave complementary Watson-Crick base pairing with the targeted DNA site.Activation of the light activated drug after the conjugate has bound thetargeted DNA site can cause cleavage of the DNA strand at the targetedDNA site. As a result, the activated drug conjugate can be used forcleavage of targeted DNA sites. The light activated conjugate can betargeted to a site on viral DNA where activation of the light activatedconjugate causes the virus to be killed. Similarly, the light activatedconjugate can be targeted to oncogenes. Other applications of targetedDNA cleavage include, but are not limited to, antisense applications,specific cleavage and subsequent recombination of DNA; destruction ofviral DNA; construction of probes for controlling gene expression at thecellular level and for diagnosis; and cleavage of DNA in footprintinganalyses, DNA sequencing, chromosome analysis, gene isolation,recombinant DNA manipulations, mapping of large genomes and chromosomes,in chemotherapy and in site directing mutagenesis.

[0118] In another preferred embodiment, the light activated drugincludes a hormone. The hormone may be targeted to a particularbiological receptor which is localized at the tissue site.

[0119] The light activated drug can be included within several mediasuitable for delivery into the body. Many light activated drugs areknown to have low water solubilities of less than 100 mg/L. As a result,achieving the desired concentration of light activated drug in anaqueous solution media for systemic delivery can often be difficult.However, localized delivery of the light activated drug requires a lowerconcentration of light activated drug within the media. As a result,when the light activated drug is delivered locally, the light activateddrug can be included in an aqueous solution.

[0120] The media can also be an emulsion which includes a lipoid as ahydrophobic phase dispersed in a hydrophilic phase. These emulsionsprovide a media which is safe for delivery into the body with aneffective concentration of light activated drug.

[0121] The media can also include microbubbles comprised from asubstrate which forms a shell. Suitable substrates for the microbubbleinclude, but are not limited to, biocompatible polymers, albumins,lipids, sugars or other substances. The light activated drug can beenclosed within the microbubble, coupled with the shell and/ordistributed in the media outside the microbubble. A preferredmicrobubble comprises a lipid substrate such as liposome. Systemicadministration of liposomes with light activated drug has been shown toresult in an increased accumulation and more prolonged retention oflight activated drugs within cultured malignant cells and within tumorsin vivo. Jori et al., Br. J. Cancer, 48:307-309 (1983); Cozzani et al.,In Porphyrins in Tumor Phototherapy, 173-183, Plenum Press (Andreoni etal. eds. 1984). As a result, inclusion of the light activated drugwithin a liposome combined with the localized delivery of the lightactivated drug can serve to enhance the localization of the lightactivated drug within the tissue site.

[0122] Including a light activated drug with the microbubbles hasnumerous advantages over microbubbles without light activated drug.After administration of microbubbles to a patient, the microbubblesoften must be ruptured to achieve their therapeutic effects. Onetechnique for rupturing microbubbles has been to expose the microbubblesto ultrasound energy. However, ultrasound energy of undesirably highintensity is frequently required to break the microbubbles. Further, theultrasound energy frequently must be matched to the resonant frequencyof the microbubbles. As a result, rupturing the microbubbles withultrasound can present numerous challenges.

[0123] Activating a light activated drug within the microbubble and/orin the substrate of the microbubble can cause the microbubble torupture. Activation of the light activated drug is believed to cause adisturbance which disrupts the shell of the microbubble enough to causethe microbubble to rupture. This disruption occurs when the lightactivated drug is coupled with the shell of the microbubble or isentirely within the microbubble. This disruption is also believed tooccur when light activated drug located the media outside themicrobubbles is activated in proximity of the microbubble. Accordingly,including a sufficient concentration light activated drug in the mediaoutside the microbubble and activating a portion of that light activateddrug can also cause rupture of the microbubbles. As a result,microbubbles can be ruptured by activating light activated drugs andwithout matching the ultrasound frequency to the resonant frequency ofthe microbubble. However, a more efficient rupturing of microbubbles canbe achieved by delivering a level of ultrasound energy which isappropriate to activate the light activated drug and which is matched tothe resonant frequency of the microbubble. Further, the cavitationthreshold can require an ultrasound intensity which is lower than theintensity required to rupture microbubbles without light activateddrugs. As a result, including light activated drug with microbubbles canreduce the intensity of ultrasound energy required to rupture themicrobubble.

[0124] The threshold value of cavitation is also reduced in theproximity of many light activated drugs. As a result, the lightactivated drug encourages cavitation in the proximity of the lightactivated drug.

[0125] The interior of the microbubbles may include a gas or may bedevoid of gas. When a gas is present, the gas can occupy any portion ofthe microbubble's volume but preferably occupies 0.01-50% of the volumeof the microbubble interior, more preferably 5-30% and most preferably10-20%. When the volume of gas is less than 0.01% of the volume,cavitation can be hindered and when the volume of gas is greater than50% the structural integrity of the microbubble shell can become tooweak for the microbubble to be transported to the tissue site. Suitablegasses for the interior of the microbubbles include, but are not limitedto, biocompatible gasses such as air, nitrogen, carbon dioxide, oxygen,argon, fluorine, xenon, neon, helium, or combinations thereof. Thepresence of tiny bubbles is known to reduce the cavitation threshold. Asa result, the presence of an appropriately sized gas bubble in themicrobubble can enhance cavitation in the proximity of the lightactivated drug.

[0126] The microbubbles are preferably 0.01-100 μm in diameter. Thissize microbubble reduces excretion of the microbubble outside the bodyand also reduces interference of the microbubble with the flow of fluidswithin the body of the patient. Further, the microbubbles preferablyhave a shell thickness of 0.001-50 μm, 0.01-5 μm and 0.1-0.5 μm. Thisthickness provides the shells with sufficient thickness that themicrobubble can withstand enough of the forces within the vasculature ofa patient to be transported through at least a portion of the patient'svasculature. Similarly, the thickness can permit the microbubbles to betransported through a lumen in an apparatus such as a catheter. However,this thickness is also sufficiently thin that alteration of theultrasound activated substance upon activation is sufficient to disruptthe shell of the microbubble and cause the microbubble to rupture.

[0127] Activating the light activated drug to rupture microbubbles cancause the light activated drug to be released from the microbubble sothe light activated drug can penetrate the tissue near the site ofrupture. Further exposure of the light activated drug to ultrasound canactivate the light activated drug within the tissue and cause death ofthe tissue as described above.

[0128] The microbubble can include a therapeutic in addition to thelight activated drug. Activation of the light activated drug can serveto rupture the microbubble and release the therapeutic from themicrobubble. As a result, the therapeutic is released in proximity to atissue site by rupturing the microbubble in proximity to the tissuesite. This is advantageous when the therapeutic can be detrimental whenadministered systemically. For instance, a therapeutic such as cisplatinis known to kill cancerous tissues but is also known to kill othertissues throughout the body. As a result, systemic administration ofcisplatin can be detrimental. However, microbubbles can serve to protecttissues from the therapeutic agent until the therapeutic agent isreleased from the carrier. For instance, when the therapeutic isenclosed within the interior of the microbubble, contact between thetherapeutic agent and tissues outside the carrier is reduced. As aresult, the carrier increases protection of tissues outside the carrierare protected from the therapeutic agent until the microbubble isruptured and the therapeutic released.

[0129] The therapeutics may be encapsulated in the microbubbles,included in the shell of the microbubbles or in the media outside themicrobubbles. By therapeutic, as used herein, it is meant an agenthaving beneficial effect on the patient.

[0130] Examples of therapeutics which can be included with themicrobubbles include, but are not limited to, hormone products such as,vasopressin and oxytocin and their derivatives, glucagon and thyroidagents as iodine products and anti-thyroid agents; cardiovascularproducts as chelating agents and mercurial diuretics and cardiacglycosides; respiratory products as xanthine derivatives (theophylline &aminophylline); anti-infectives as aminoglycosides, antifingals(amphotericin), penicillin and cephalosporin antibiotics, antiviralagents as Zidovudine, Ribavirin, Amantadine, Vidarabine, and Acyclovir,antihelmintics, antimalarials, and antituberculous drugs; biologicals asimmune serums, antitoxins and antivenins, rabies prophylaxis products,bacterial vaccines, viral vaccines, toxoids; antineoplasticsasnitrosureas, nitrogen mustards, antimetabolites (fluorouracil,hormones, asprogesings and estrogens and antiestrogens; antibiotics asDactinomycin; mitotic inhibitors as Etoposide and the Vinca alkaloids,Radiopharmaceuticals as radioactive iodine and phosphorus products; aswell as Interferon, hydroxyurea, procarbazine, Dacarbazine, Mitotane,Asparaginase and cyclosporins.

[0131] Other suitable therapeutics include, but are not limited to:thrombolytic agents such as urokinase; coagulants such as thrombin;antineoplastic agents, such as platinum compounds (e.g., spiroplatin,cisplatin, and carboplatin), methotrexate, adriamycin, taxol, mitomycin,ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adsnine,mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan(e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine, mitotane,procarbazine hydrochloride dactinomycin (actinomycin D),daunorubicinhydrochloride, doxorubicin hydrochloride, mitomycin,plicamycin (mithramycin), aminoglutethimide, estramustine phosphatesodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifencitrate, testolactone, trilostane, amsacrine (m-AMSA), asparaginase(L-asparaginase) Erwinaasparaginase, etoposide (VP-16), interferonalpha-2a, interferon alpha-2b, teniposide (VM-26), vinblastine sulfate(VLB), vincristine sulfate, bleomycin, bleomycin sulfate, methotrexate,adriamycin, and arabinosyl; blood products such as parenteral iron,hemin; biological response modifiers such as muramyldipeptide,muramyltripeptide, microbial cell wall components, lymphokines (e.g.,bacterial endotoxin such as lipopolysaccharide, macrophageactivationfactor), sub-units of bacteria (such as Mycobacteria,Corynebacteria), the synthetic dipeptideN-acetyl-muramyl-L-alanyl-D-isoglutamine; anti-fungalagents such asketoconazole, nystatin, griseofulvin, flucytosine (5-fc), miconazole,amphotericin B, ricin, and beta-lactam antibiotics (e.g., sulfazecin);hormones such as growth hormone, melanocyte stimulating hormone,estradiol, beclomethasone dipropionate, betamethasone, betamethasoneacetate and betamethasone sodium phosphate,vetamethasonedisodiumphosphate, vetamethasone sodium phosphate,cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasonesodium phosphate, flunsolide, hydrocortisone, hydrocortisone acetate,hydrocortisonecypionate, hydrocortisone sodium phosphate, hydrocortisonesodium succinate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, paramethasone acetate,prednisolone, prednisoloneacetate, prednisolone sodium phosphate,prednisolone rebutate, prednisone, triamcinolone, triamcinoloneacetonide, triamcinolone diacetate, triamcinolone hexacetonide andfludrocortisone acetate; vitamins such ascyanocobalamin neinoic acid,retinoids and derivatives such as retinolpalmitate, andalpha-tocopherol; peptides, such as manganese super oxidedimutase;enzymes such as alkaline phosphatase; anti-allergic agents such asamelexanox; anti-coagulation agents such as phenprocoumon and heparin;circulatory drugs such as propranolol; metabolic potentiators suchasglutathione; antituberculars such as para-aminosalicylic acid,isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochlorideethionamide, pyrazinamide, rifampin, and streptomycin sulfate;antivirals such as acyclovir, amantadine azidothymidine (AZT orZidovudine), Ribavirin andvidarabine monohydrate (adenine arabinoside,ara-A); antianginals such asdiltiazem, nifedipine, verapamil, erythrityltetranitrate, isosorbidedinitrate, nitroglycerin (glyceryl trinitrate)and pentaerythritoltetranitrate; anticoagulants such as phenprocoumon,heparin; antibiotics such as dapsone, chloramphenicol, neomycin,cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin rifampinand tetracycline; antiinflammatories such as difunisal, ibuprofen,indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates;antiprotozoans such as chloroquine, hydroxychloroquine, metronidazole,quinine and meglumine antimonate; antirheumatics such as penicillamine;narcotics such as paregoric; opiates such as codeine, heroin, methadone,morphine and opium; cardiac glycosides such as deslanoside, digitoxin,digoxin, digitalin and digitalis; neuromuscular blockers such asatracurium besylate, gallamine triethiodide, hexafluorenium bromide,metocurine iodide, pancuronium bromide, succinylcholine chloride(suxamethonium chloride), tubocurarine chloride and vecuronium bromide;sedatives (hypnotics) such as amobarbital, amobarbital sodium,aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol,ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazinehydrochloride, methyprylon, midazolam hydrochloride, paraldehyde,pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbitalsodium, talbutal, temazepam and triazolam; local anesthetics such asbupivacaine hydrochloride, chloroprocaine hydrochloride,etidocainehydrochloride, lidocaine hydrochloride, mepivacainehydrochloride, procainehydrochloride and tetracaine hydrochloride;general anesthetics such asdroperidol, etomidate, fentanyl citrate withdroperidol, ketaminehydrochloride, methohexital sodium and thiopentalsodium; and radioactive particles or ions such as strontium, iodiderhenium and yttrium.

[0132] In certain preferred embodiments, the therapeutic is a monoclonalantibody, such as a monoclonal antibody capable of binding to melanomaantigen.

[0133] Other preferred therapeutics include genetic material such asnucleic acids, RNA, and DNA, of either natural or synthetic origin,including recombinant RNA and DNA and antisense RNA and DNA. Types ofgenetic material that may be used include, for example, genes carried onexpression vectors such as plasmids, phagemids, cosmids, yeastartificial chromosomes (YACs), and defective or “helper” viruses,antigene nucleic acids, both single and double stranded RNA and DNA andanalogs thereof, such asphosphorothioate and phosphorodithioateoligodeoxynucleotides. Additionally, the genetic material may becombined, for example, with proteins or other polymers.

[0134] Examples of genetic therapeutics that may be included in themicrobubbles include DNA encoding at least a portion of an HLAgene, DNAencoding at least a portion of dystrophin, DNA encoding at least aportion of CFTR, DNA encoding at least a portion of IL-2, DNA encodingat least a portion of TNF, an antisense oligonucleotide capable ofbinding the DNA encoding at least a portion of Ras.

[0135] DNA encoding certain proteins may be used in the treatment ofmany different types of diseases. For example, adenosine deaminase maybe provided to treat ADA deficiency; tumor necrosis factor and/orinterleukin-2 may be provided to treat advanced cancers; HDL receptormay be provided to treat liver disease; thymidine kinase may be providedto treat ovarian cancer, brain tumors, or HIV infection; HLA-D7 may beprovided to treat malignant melanoma; interleukin-2 may be provided totreat neuroblastoma, malignant melanoma, or kidney cancer; interleukin-4may be provided to treat cancer; HIV env may be provided to treat HIVinfection; antisense ras/p53 may be provided to treat lung cancer; andFactor VIII may be provided to treat Hemophilia B. See, for example,Science 258, 744-746.

[0136] If desired, more than one therapeutic may be included in themedia. For example, a single microbubble may contain more than onetherapeutic or microbubbles containing different therapeutics may beco-administered. By way of example, a monoclonal antibody capable ofbinding to melanoma antigen and an oligonucleotide encoding at least aportion of IL-2 may be administered in a single microbubble. The phrase“at least a portion of,” as used herein, means that the entire gene neednot be represented by the oligonucleotide, so long as the portion of thegene represented provides an effective block to gene expression.Further, microbubbles including a therapeutic can be administeredbefore, after, during or intermittently with the administration ofmicrobubbles without a therapeutic. For instance, microbubbles without atherapeutic and microbubbles including a coagulant such as thrombin canbe administered to a patient having liver cancer. Activating the lightactivated drug included with the microbubbles serves to rupture themicrobubbles and release the light activated drug and thrombin from themicrobubbles. Further activation of the light activated drug can causetissue death and the thrombin can cause coagulation in and around thedamaged tissues.

[0137] Prodrugs may be included in the microbubbles, and are includedwithin the ambit of the term therapeutic, as used herein. Prodrugs arewell known in the art and include inactive drug precursors which, whenexposed to high temperature, metabolizing enzymes, cavitation and/orpressure, in the presence of oxygen or otherwise, or when released fromthe microbubbles, will form active drugs. Such prodrugs can be activatedvia the application of ultrasound to the prodrug-containing microbubbleswith the resultant cavitation, heating, pressure, and/or release fromthe microbubbles. Suitable prodrugs will be apparent to those skilled inthe art, and are described, for example, in Sinkula et al., J. Pharm.Sci. 1975 64, 181-210, the disclosure of which is hereby incorporatedherein by reference in its entirety. Prodrugs, for example, may compriseinactive forms of the active drugs wherein a chemical group is presenton the prodrug which renders it inactive and/or confers solubility orsome other property to the drug. In this form, the prodrugs aregenerally inactive, but once the chemical group has been cleaved fromthe prodrug, by heat, cavitation, pressure, and/or by enzymes in thesurrounding environment or otherwise, the active drug is generated. Suchprodrugs are well described in the art, and comprise a wide variety ofdrugs bound to chemical groups through bonds such as esters to short,medium or long chain aliphatic carbonates, hemiesters of organicphosphate, pyrophosphate, sulfate, amides, amino acids, azo bonds,carbamate, phosphamide, glucosiduronate, N-acetylglucosamine andbeta-glucoside. Examples of drugs with the parent molecule and thereversible modification or linkage are as follows: convallatoxin withketals, hydantoin with alkyl esters, chlorphenesin with glycine oralanins esters, acetaminophen with caffeine complex, acetylsalicylicacid with THAM salt, acetylsalicylic acid with acetamidophenyl ester,naloxone with sulfateester, 15-methylprostaglandin F sub 2 with methylester, procaine with polyethylene glycol, erythromycin with alkylesters, clindamycin with alkylesters or phosphate esters, tetracyclinewith betains salts, 7-acylaminocephalosporins with ring-substitutedacyloxybenzyl esters, nandrolone with phenylproprionate decanoateesters, estradiol with enolether acetal, methylprednisolone with acetateesters, testosterone with n-acetylglucosaminide glucosiduronate(trimethylsilyl) ether, cortisol or prednisolone or dexamethasone with21-phosphate esters. Prodrugs may also be designed as reversible drugderivatives and utilized as modifiers to enhance drug transport tosite-specific tissues. Examples of parent molecules with reversiblemodifications or linkages to influence transport to a site specifictissue and for enhanced therapeutic effect include isocyanate withhaloalkyl nitrosurea, testosterone with propionateester, methotrexate(3-5′-dichloromethotrexate) with dialkyl esters, cytosine arabinosidewith 5′-acylate, nitrogen mustard (2,2′-dichloro-N-methyldiethylamine),nitrogen mustard with aminomethyltetracycline, nitrogen mustard withcholesterol or estradiol ordehydroepiandrosterone esters and nitrogenmustard with azobenzene. As one skilled in the art would recognize, aparticular chemical group to modify a given drug may be selected toinfluence the partitioning of the drug into either the shell or theinterior of the microbubbles. The bond selected to link the chemicalgroup to the drug may be selected to have the desired rate ofmetabolism, e.g., hydrolysis in the case of ester bonds in the presenceof serum esterases after release from the microbubbles. Additionally,the particular chemical group may be selected to influence thebiodistribution of the drug employed in the microbubbles, e.g.,N,N-bis(2-chloroethyl)-phosphorodiamidicacid with cyclic phosphoramidefor ovarian adenocarcinoma. Additionally, the prodrugs employed withinthe microbubbles may be designed to contain reversible derivatives whichare utilized as modifiers of duration of activity to provide, prolong ordepot action effects. For example, nicotinic acid may be modified withdextran and carboxymethlydextran esters, streptomycin with alginic acidsalt, dihydrostreptomycin with pamoate salt, cytarabine (ara-C) with5′-adamantoats ester, ara-adenosine (ara-A) with 5-palmirate and5′-benzoate esters, amphotericin B with methyl esters, testosterone with17-beta-alkyl esters, estradiol with formate ester, prostaglandin with2-(4-imidazolyl) ethylamine salt, dopamine with amino acid amides,chloramphenicol with mono- and bis(trimethylsilyl) ethers, andcycloguanil with pamoate salt. In this form, a depot or reservoir oflong-acting drug may be released in vivo from the prodrug bearingmicrobubbles. In addition, compounds which are generally thermallylabile may be utilized to create toxic free radical compounds. Compoundswith azolinkages, peroxides and disulfide linkages which decompose withhigh temperature are preferred. With this form of prodrug, azo, peroxideor disulfide bond containing compounds are activated by cavitationand/or increased heating caused by the interaction of ultra with themicrobubbles to create cascades of free radicals from these prodrugsentrapped therein. A wide variety of drugs or chemicals may constitutethese prodrugs, such as azo compounds, the general structure of suchcompounds being R—N═N—R, wherein R is a hydrocarbon chain, where thedouble bond between the two nitrogen atoms may react to create freeradical products in vivo. Exemplary drugs or compounds which may be usedto create free radical products include azo containing compounds such asazobenzene,2,2′-azobisisobutyronitrile, azodicarbonamide, azolitmin,azomycin, azosemide, azosulfamide, azoxybenzene, aztreonam, sudan III,sulfachrysoidine, sulfamidochrysoidine and sulfasalazine, compoundscontaining disulfide bonds such as sulbentine, thiamine disulfide,thiolutin, thiram, compounds containing peroxides such as hydrogenperoxide and benzoylperoxide, 2,2′-azobisisobutyronitrile,2,2′-azobis(2-amidopropane) dihydrochloride, and2,2′-azobis(2,4-dimethylvaleronitrile). A microbubble having oxygen gason its interior should create extensive free radicals with cavitation.Also, metal ions from the transition series, especially manganese, ironand copper can increase the rate of formation of reactive oxygenintermediates from oxygen. By including metal ions within themicrobubbles, the formation of free radicals in vivo can be increased.These metal ions may be incorporated into the microbubbles as freesalts,as complexes, e.g., with EDTA, DTPA, DOTA or desferrioxamine, orasoxides of the metal ions. Additionally, derivatized complexes of themetal ions may be bound to lipid head groups, or lipophilic complexes ofthe ions may be incorporated into a lipid bilayer, for example. Whenexposed to thermal stimulation, e.g., cavitation, these metal ions thenwill increase the rate of formation of reactive oxygen intermediates.Further, radiosensitizers such as metronidazole and misonidazole may beincorporated into the gas-filled liposomes to create free radicals onthermal stimulation. By way of an example of the use of prodrugs, anacylated chemical group may be bound to a drug via an ester linkagewhich would readily cleave in vivo by enzymatic action in serum. Theacylated prodrug can be included in the microbubble. When themicrobubble is ruptured, the prodrug will then be exposed to the serum.The ester linkage is then cleaved by esterases in the serum, therebygenerating the drug. Similarly, ultrasound may be utilized not only toactivate the light activated drug so as to burst the gas-filledliposome, but also to cause thermal effects which may increase the rateof the chemical cleavage and the release of the active drug from theprodrug. The microbubbles may also be designed so that there is asymmetric or an asymmetric distribution of the therapeutic both insideand outside of the microbubble. The particular chemical structure of thetherapeutics may be selected or modified to achieve desired solubilitysuch that the therapeutic may either be encapsulated within the interiorof the microbubble or couple with the shell of the microbubble. Theshell-bound therapeutic may bear one or more acyl chains such that, whenthe microbubble is popped or heated or ruptured via cavitation, theacylated therapeutic may then leave the surface and/or the therapeuticmay be cleaved from the acyl chains chemical group. Similarly, othertherapeutics may be formulated with a hydrophobic group which isaromatic or sterol in structure to incorporate into the surface of themicrobubble.

[0138] When the microbubble is a liposome, the liposomes can be “fastbreaking”. In fast breaking liposomes, the light activated drug-liposomecombination is stable in vitro but, when administered in vivo, the lightactivated drug is rapidly released into the bloodstream where it canassociate with serum lipoproteins. As a result, the localized deliveryof liposomes combined with the fast breaking nature of the liposomes canresult in localization of the light activated drug and/or thetherapeutic in the tissues near the catheter. Further, the fast breakingliposomes can prevent the liposomes from leaving the vicinity of thecatheter intact and then concentrating in non-targeted tissues such asthe liver. Delivery of ultrasound energy from the catheter can alsoserve to break apart the liposomes after they have been delivered fromthe catheter.

[0139] A catheter for locally delivering a media including a lightactivated drug includes an elongated body with at least one utilitylumen extending through the elongated body. The utility lumens can beused to deliver the media including the light activated drug locally toa tissue site and/or to receive a guidewire so the catheter can beguided to the tissue site. The ultrasound assembly can include anultrasound transducer designed to transmit ultrasound energy whichactivates the light activated drug.

[0140] A support member can support the ultrasound transducer adjacentto an outer surface of the elongated body so as to define a chamberbetween the ultrasound transducer and the elongated body. The chambercan be filled with a material which creates a low acoustic impedance toreduce the exposure of at least one utility lumen within the elongatedbody to ultrasound energy delivered from the ultrasound transducer. Forinstance, the chamber can be filled with a material which absorbs,reflects or prevents transmission of ultrasound energy through thechamber. Alternatively, the chamber can be evacuated to reducetransmission of ultrasound energy through the chamber. Reducing theexposure of at least one lumen to the ultrasound energy reduces exposureof media delivered through the at least one lumen to the ultrasoundenergy. As a result, the effect of the ultrasound energy on the lightactivated drug is reduced until the light activated drug has beendelivered out of the catheter. Further, ultrasound energy is known torupture microbubbles. As a result, when the media includes microbubbles,the chamber reduces the opportunity for the ultrasound energy to rupturethe microbubbles within the catheter.

[0141] The support member can have ends which extend beyond theultrasound member. As a result, the chamber can be positioned adjacentto the entire longitudinal length of the ultrasound transducer and canextend beyond the ends of the ultrasound transducer. This configurationmaximizes the portion of the ultrasound transducer which is adjacent tothe chamber. Increasing the portion of ultrasound transducer adjacent tothe chamber can reduce the amount of ultrasound energy transmitted tothe utility lumens. The ultrasound assembly can include an outer coatingover the ultrasound transducer. Temperature sensors can be positioned inthe outer coating adjacent to ultrasound transducer. This position ofthe temperature sensors feedback regarding the temperature adjacent tothe ultrasound transducers where the thermal energy has a reducedopportunity to dissipate. As a result, the temperature sensors provide ameasure of the temperature on the exterior surface of the transducer.

[0142]FIGS. 1A-1B illustrates a catheter 10 for delivering a mediaincluding a light activated drug to a tissue site. The catheter 10includes an ultrasound assembly 12 for delivering ultrasound energy tolight activated drug within the tissue site. The catheter 10 includes anelongated body 14 with a utility lumen 16 extending through theelongated body 14. The utility lumen 16 can receive a guidewire (notshown) so the catheter 10 can be threaded along the guidewire. Theutility lumen 16 can also be used for the delivering media which includea light activated drug. A fiber optic can also be positioned in theutility lumen 16 to provide a view of the tissue site or to providelight to the tissue site. As a result, the catheter can also be used asan endoscope.

[0143] The ultrasound assembly 12 can also include an outer coating 18.Suitable outer coatings 18 include, but are not limited to, polyimide,parylene and polyester. An ultrasound transducer 20 is positioned withinthe outer coating 18. Suitable ultrasound transducers 20 include, butare not limited to, PZT-4D, PZT-4, PZT-8 and cylindrically shapedpiezoceramics. When the ultrasound transducer 20 has a cylindricalshape, the ultrasound transducer 20 can encircle the elongated body 14as illustrated in FIG. 1C. One or more temperature sensors 22 can bepositioned in the outer coating 18. The temperature sensors 22 can bepositioned adjacent to the ultrasound transducer 20 to provide feedbackregarding the temperature adjacent to the ultrasound transducer 20. Thetemperature sensors can be in electrical communication with anelectrical coupling 24. The electrical coupling 24 can be coupled with afeedback control system (not shown) which adjusts the level of theultrasound energy delivered from the ultrasound transducer 20 inresponse to the temperature at the temperature sensors 22.

[0144] The catheter 10 can include a perfusion lumen 25. The perfusionlumen 25 allows fluid to flow from outside the catheter into the utilitylumen 16. Once a guidewire has been removed from the utility lumen 16,fluid flow which is obstructed by the ultrasound assembly can continuethrough the perfusion lumen 25 and the utility lumen. As illustrated inFIGS. 2A-2B, the ultrasound assembly 12 can be flush with the elongatedbody 14. Further, the ultrasound transducer 20 and the temperaturesensors 22 can be positioned within the elongated body 14. Thisconfiguration of elongated body 14 and ultrasound transducer 20 caneliminate the need for the outer coating 18 illustrated in FIGS. 1A-1C.

[0145] As illustrated in FIG. 3A, the catheter 10 can also include amedia delivery port 26, a media inlet port 28 and a second utility lumen16A. The media inlet port 28 is designed to be coupled with a mediasource (not shown). Media can be transported from the media source andthrough the media delivery port 26 via the second utility lumen 16A. Asa result, a guidewire can be left within the utility lumen 16 whilemedia is delivered via the second utility lumen 16A.

[0146]FIG. 4A illustrates a catheter 10 including a plurality ofultrasound assemblies 12. FIGS. 4B-4C are cross sections of a catheter10 with a second utility lumen 16A coupled with the media delivery ports26. The second utility lumen 16A can also be coupled with the mediainlet port 28 illustrated in FIG. 4A. The media inlet port 28 isdesigned to be coupled with a media source (not shown). Media can betransported from the media source and through the media delivery ports26 via the second utility lumen 16A.

[0147] The catheter 10 can include a balloon 30 as illustrated in FIG.5A. The balloon 30 can be constructed from an impermeable material or apermeable membrane or a selectively permeable membrane which allowscertain media to flow through the membrane while preventing other mediafrom flowing through the membrane. Suitable membranous materials for theballoon 30 include, but are not limited to cellulose, cellulose acetate,polyvinylchloride, polyolefin, polyurethane and polysulfone. When theballoon 30 is constructed from a permeable membrane or a selectivelypermeable membrane, the membrane pore sizes are preferably 5 A-2 μm,more preferably 50 A-900 A and most preferably 100 A-300 A in diameter.

[0148] As illustrated in FIG. 5B, an ultrasound assembly 12, a firstmedia delivery port 26A and a second media delivery port 26B can bepositioned within the balloon 30. The first and second media deliveryports 26A, 26B are coupled with a second utility lumen 16A and thirdutility lumen 16B. The second and third utility lumens 16A, 16B can becoupled with the same media inlet port 28 or with independent mediainlet ports 28. When the first and second media delivery ports 26A, 26Bare coupled with different media inlet ports 28, different media can bedelivered via the second and third media delivery ports 26A, 26B. Forinstance, a medication media can be delivered via the third utilitylumen 16B and an expansion media can be delivered via the second utilitylumen 16A. The medication media can include drugs or other medicamentswhich can provide a therapeutic effect. The expansion media can serve toexpand the balloon 30 or wet the membrane comprising the balloon 30.Wetting the membrane comprising the balloon 30 can cause a minimallypermeable membrane to become permeable.

[0149] The ultrasound assembly 12 can be positioned outside the balloon30 as illustrated in FIGS. 6A-6C. In FIG. 6A the balloon 30 ispositioned distally of the ultrasound assembly 12 and in FIG. 6B theultrasound assembly 12 is positioned distally of the balloon 30. FIG. 6Cis a cross section a catheter 10 with an ultrasound assembly 12positioned outside the balloon 30. The catheter includes a secondutility lumen 16A coupled with a first media delivery port 26A. Thesecond utility lumen 16A can be used to deliver an expansion mediaand/or a medication media to the balloon 30. When the balloon 30 isconstructed from a permeable membrane, the medication media and/or theexpansion media can pass through the balloon 30. Similarly, when theballoon 30 is constructed from a selectively permeable membrane,particular components of the medication media and/or the expansion mediacan pass through the balloon 30. Pressure can be used to drive the mediaor components of the media across the balloon 30. Other means such asphoresis can also be used to drive the media or components of the mediaacross the balloon 30.

[0150] As illustrated in FIG. 6C, the ultrasound assembly 12 may bepositioned at the distal end of the catheter 10. The second utilitylumen 16A can be used to deliver an expansion media and/or a medicationmedia to the balloon 30. The utility lumen 16 can be used to deliver amedication media as well as to guide the catheter 10 along a guidewire.

[0151] As illustrated in FIGS. 7A-7C, the catheter 10 can include asecond media delivery port 26B positioned outside the balloon. In FIGS.7A-7C the ultrasound assembly 12 and the second media delivery port 26Bare positioned distally relative to a balloon 30, however, the balloon30 can be positioned distally relative to the ultrasound assembly 12 andthe second media delivery port 26B. In FIG. 7A the ultrasound assembly12 is positioned distally of the second media delivery port 26B and inFIG. 7B the second media delivery port 26B is positioned distally of theultrasound assembly 12.

[0152]FIG. 7C is a cross section of the catheter 10 illustrated in FIG.7A. The catheter 10 includes first and second media delivery ports 26A,26B coupled with a second utility lumen 16A and third utility lumen 16B.The second and third utility lumens 16A, 16B can be coupled withindependent media inlet ports 28 (not shown). The second utility lumen16A can be used to deliver an expansion media and/or a medication mediato the balloon 30 while the third utility lumen 16B can be used todeliver a medication media through the second media delivery port 26B.

[0153] As illustrated in FIGS. 8A-8B, the catheter 10 can include afirst balloon 30A and a second balloon 30B. The ultrasound assembly 12can be positioned between the first and second balloons 30A, 30B. Asecond media delivery port 26B can optionally be positioned between thefirst and second balloons 30A, 30B. In FIG. 8A the second media deliveryport 26B is positioned distally relative to the ultrasound assembly andin FIG. 8B the ultrasound assembly is positioned distally relative tothe second media delivery port 26B.

[0154]FIG. 8C is a cross section of the first balloon 30A illustrated inFIG. 8B. The catheter includes a second, third and fourth utility lumens16A, 16B, 16C. The second utility lumen 16A is coupled with a firstmedia delivery port 26A within the first balloon. The third utilitylumen 16B is coupled with the second media delivery port 26B and thefourth utility lumen 16C is coupled with a third media delivery port 26Cin the second balloon 30B (not shown). The second and fourth utilitylumens 16A, 16C can be used to deliver expansion media and/or medicationmedia to the first and second balloon 30A, 30B. The second and fourthutility lumens 16A, 16C can be coupled with the same media inlet port orwith independent media inlet ports (not shown). When the second andfourth utility lumens are coupled with the same media inlet port, thepressure within the first and second balloons 30A, 30B will be similar.When the second and fourth utility lumens are coupled with independentmedia inlet ports, different pressures can be created within the firstand second balloons 30A, 30B. The third utility lumen 16B can be coupledwith an independent media inlet port and can be used to deliver amedication media via the second media delivery port 26B.

[0155]FIGS. 9A-9I illustrate operation of various embodiments ofcatheters 10 for delivering ultrasound energy to a light activated drugwithin a tissue site. FIGS. 9A-9I illustrate the tissue site 32 as anatheroma in a vessel 34, however, it is contemplated that the catheter10 can be used with other tissue sites 32 such as a tumor and that thecatheter 10 can be positioned within the vasculature of the tumor. Ineach of FIGS. 9A-9I, the catheter 10 is illustrated as being within avessel 34. The catheter 10 can be positioned within the vessel 34 byapplying conventional over-the-guidewire techniques and can be verifiedby including radiopaque markers upon the catheter 10.

[0156] In FIG. 9A, the catheter 10 is positioned so the ultrasoundassembly 12 is adjacent to a tissue site 32 within a vessel 34. When thecatheter 10 is in position, the guidewire is removed from the utilitylumen 16 and media can be delivered via the utility lumen 16 asillustrated by the arrows 36. In FIG. 9A, the media includesmicrobubbles 38 but can alternatively be an emulsion. The media isdelivered to the tissue site 32 via the utility lumen 16 and ultrasoundenergy 40 is delivered from the ultrasound assembly 12. Suitable periodsfor delivering the ultrasound energy include, but are not limited to, 1minute to three hours, 2 minutes to one hour and 10-30 minutes.

[0157] Suitable intensities for the ultrasound energy include, but arenot limited to, 0.1-1000 W/cm², 1-100 W/cm² and 10-50 W/cm². Suitablefrequencies for the ultrasound energy include, but are not limited to,10 kHz-100 MHz and 10 kHz-50 MHz but is preferably 20 kHz-10 MHz.Suitable ultrasound energies also include, but are not limited to 0.02to 10 w/cm² at a frequency of about 20 KHz to about 10 MHz and morepreferably about 0.3 W/cm² at a frequency of about 1.3 MHz. Theultrasound energy can be intermittently switched between a first andsecond frequency to increase the efficiency of microbubble rupture andto increase activation of the light activated drug. For instance, theultrasound energy can be switched between about 100 kHz and about 270kHz in short pulses of approximately 0.001-10 seconds duration.Similarly, the ultrasound energy can be switched between first andsecond intensities. When the catheter includes a plurality of ultrasoundtransducers as will be discussed below, the first and second frequenciescan be provided by different ultrasound transducers. Similarly, thefirst and second intensities can be provided by different ultrasoundtransducers. Further, when the catheter includes a plurality ofultrasound transducers each transducer can simultaneously transmitultrasound energy with different intensity and/or frequency.

[0158] The delivery of ultrasound energy 40 can be before, after, duringor intermittently with the delivery of the microbubbles 38. As discussedabove, the microbubbles 38 can be “fast breaking” so they rupture uponexiting the utility lumen and being exposed to the vessel 34. Asdescribed above, the ultrasound energy from the ultrasound assembly 12can cause the microbubbles 38 within the delivered media to rupture. Aswill be described in more detail below, the ultrasound assembly can bedesigned to reduce the exposure of media within the catheter 10 to theultrasound energy from the ultrasound assembly 12. When the catheter 10is so designed, the number of microbubbles 38 which rupture within thecatheter is reduced and the number of microbubbles 38 which ruptureoutside the catheter is increased.

[0159] Delivery of the ultrasound energy before delivery of the lightactivated drug can enhance absorption of the light activated drug intothe tissue site. Delivery of the ultrasound energy a pre-determined timeafter delivery of the light activated drug can provide the lightactivated drug time to penetrate the tissue site. The pre-determinedtime can be of sufficient duration that at least a portion of the lightactivated drug penetrates into the tissue site. The pre-determined timecan also be of sufficient duration that the light activated druglocalizes within the lipid rich tissue of the atheroma. Sufficient timebetween delivery of the media and the ultrasound energy include but arenot limited to, 1 minute to 48 hours, 1 minute to 3 hours, 1 to 15minutes and 1 to 2 minutes. Once the light activated drug has penetratedthe tissue site 32, the ultrasound energy from the ultrasound assembly12 can activate the light activated dug within the tissue site 32 so asto cause tissue death within the tissue site 32.

[0160] In FIG. 9B, ultrasound energy 40 is delivered from the ultrasoundtransducer 20 and a media is delivered through the media delivery port26 as illustrated by the arrows 36. The delivery of ultrasound energy 40can be before, after, during or intermittently with the delivery of themedia via the media delivery port 26. As illustrated in FIG. 9C, theguidewire 104 can remain in the utility lumen 16 during the delivery ofthe media via the media delivery ports 26. As will be discussed infurther detail below, the ultrasound assembly can be designed to reducethe transmission of the ultrasound energy into the utility lumen.Because the transmission of ultrasound energy 40 into the utility lumen16 is reduced, the change in the frequency of the ultrasound transducer20 which is due to the presence of the guidewire in the utility lumen 16is also reduced.

[0161] In FIG. 9D, a catheter 10 including a balloon 30 is positionedwith the balloon adjacent to the tissue site 32. In FIG. 9E, the balloon30 is expanded into contact with the tissue site 32. As discussed above,the catheter 10 can include a perfusion lumen which permits a continuousflow of fluid from the vessel through the utility lumen during thepartial or full obstruction of the vessel by the balloon. When theballoon 30 is constructed from a membrane or a selectively permeablemembrane a media can be delivered to the tissue site 32 via the balloon30. The media can serve to wet the membrane or can include a drug orother medicament which provides a therapeutic effect. Ultrasound energy40 can be delivered from the ultrasound assembly 12 before, after,during or intermittently with the delivery of the media. The ultrasoundenergy 40 can serve to drive the media across the membrane viaphonophoresis or can enhance the therapeutic effect of the media.

[0162] In FIG. 9F a catheter 10 with an ultrasound assembly 12 outside aballoon 30 is positioned at the tissue site 32 so the ultrasoundassembly 12 is adjacent to the tissue site 32. A fluid within the vesselflows past the balloon as indicated by the arrow 42. In FIG. 9G, theballoon 30 is expanded into contact with the vessel 34. The balloon 30can be constructed from an impermeable material so the vessel 34 isoccluded. As a result, the fluid flow through the vessel 34 is reducedor stopped. A medication media is delivered through the utility lumen 16and ultrasound energy 40 is delivered from the ultrasound assembly 12.In embodiments of the catheter 10 including a media delivery port 26outside of the balloon 30 (i.e. FIGS. 7A-7C), the medication media canbe delivered via the media delivery port 26. Further, a first medicationmedia can be delivered via the media delivery port 26 while a secondmedication media can be delivered via the utility lumen 16 or while aguidewire is positioned within the utility lumen 16. The ultrasoundenergy 40 can be delivered from the ultrasound assembly 12 before,after, during or intermittently with the delivery of the media. Theocclusion of the vessel 34 before the delivery of the media can serve toprevent the media from being swept from the tissue site 32 by the fluidflow. Although the balloon 30 illustrated in FIGS. 9F-9G is positionedproximally relative to the ultrasound assembly 12, the fluid flowthrough the vessel 34 can also be reduced by expanding a single balloon30 which is positioned distally relative to the ultrasound assembly 12.

[0163] In FIG. 9H a catheter 10 including a first balloon 30A and asecond balloon 30B is positioned at a tissue site 32 so the ultrasoundassembly 12 is positioned adjacent to the tissue site 32. A fluid withinthe vessel 34 flows past the balloon 30 as indicated by the arrow 42. InFIG. 9I, the first and second balloons 30A, 30B are expanded intocontact with the vessel 34. The first and second balloons 30A, 30B canbe constructed from an impermeable material so the vessel 34 is occludedproximally and distally of the ultrasound assembly 12. As a result, thefluid flow adjacent to the tissue site 32 is reduced or stopped. Amedication media is delivered through the media delivery port 26 andultrasound energy 40 is delivered from the ultrasound assembly 12. Theultrasound energy 40 can be delivered from the ultrasound assembly 12before, after, during or intermittently with the delivery of the media.The occlusion of the vessel 34 before the delivery of the media canserve to prevent the media from being swept from the tissue site 32 bythe fluid flow.

[0164] In each of the FIGS. 9A-9I illustrated above, the media can besystemically delivered. The catheter 10 is positioned adjacent to thetissue site before, after or during the systemic administration of themedia. When the media includes microbubbles which must be burst beforetheir therapeutic effect can be obtained, the ultrasound energy can bedelivered after the microbubbles have had sufficient time to reach thedesired tissue site in sufficient concentrations. A level of ultrasoundwhich ruptures the microbubbles is then delivered from the ultrasoundassembly. After rupture of the microbubbles, the delivery of ultrasoundenergy can be stopped to provide the light activated drug or othertherapeutic time to penetrate the tissue site. The delivery of theultrasound energy can also be continuous to maximize the number ofmicrobubbles which are burst.

[0165] When the media is systemically delivered and the light activateddrug is included in media which does not require an ultrasound activatedrelease, the behavior of the light activated drug within the patientmust be taken into consideration. As described above, many light drugssuch as the macrocycles, initially disperse throughout the body andwhere they are taken up by most tissues. After a period of time, usuallybetween 3 and 48 hours, the drug clears from most normal tissue and isretained to a greater degree in lipid rich regions such as the liver,kidney, tumor and atheroma. As a result, when the tissue site is not alipid rich region, the ultrasound energy should be delivered to thetissue site within 3 to 48 hours of systemically administering themedia. However, when the tissue site is lipid rich, improved results canbe achieved by waiting 3 to 48 hours after systemic administration ofthe media before delivering the ultrasound energy.

[0166]FIG. 10A provides a cross section of an ultrasound assembly whichreduces transmission of ultrasound energy from the ultrasound transducerinto the catheter. The ultrasound assembly 12 includes a support member44. Suitable support members 44 include, but are not limited to,polyimide, polyester and nylon. The support member 44 can be attached tothe ultrasound transducer 20. Suitable means for attaching theultrasound transducer 20 to the support member 44 include, but are notlimited to, adhesive bonding and thermal bonding.

[0167] The support member 44 supports the ultrasound member 44 at anexternal surface 46 of the elongated body 14 such that a chamber 48 isdefined between the ultrasound transducer 20 and the external surface 46of the elongated body 14. The chamber 48 preferably has a height from0.25-10 μm, more preferably from 0.50-5 μm and most preferably from0.0-1.5 μm. The support member 44 can be supported by supports 50positioned at the ends 52 of the support member 44 as illustrated inFIG. 10B. The supports 50 can be integral with the support member 44 asillustrated in FIG. 10C. The outer coating 18 can serve as the supportsas illustrated in FIG. 10D.

[0168] The ends 52 of the support member 44 can extend beyond the ends54 of the ultrasound transducer 20. The supports 50 can be positionedbeyond the ends 54 of the ultrasound transducer 20. As a result, thechamber 48 can extend along the longitudinal length 56 of the ultrasoundtransducer 20, maximizing the portion of the ultrasound transducer 20which is adjacent to the chamber 48. The chamber 48 can be filled with amedium which absorbs ultrasound energy or which prevents transmission ofultrasound energy. Suitable gaseous media for filling the chamber 48include, but are not limited to, helium, argon, air and nitrogen.Suitable solid media for filling the chamber 48 include, but are notlimited to, silicon and rubber. The chamber 48 can also be evacuated.Suitable pressures for an evacuated chamber 48 include, but are notlimited to, negative pressures to −760 mm Hg.

[0169] The ultrasound assembly can include a second ultrasoundtransducer 20A as illustrated in FIGS. 11A-11H. In FIGS. 11A-11C oneultrasound transducer encircles the other and in FIGS. 11D-11H theultrasound transducers are longitudinally adjacent to one another. Theultrasound transducers 20, 20A can be in contact with one another asillustrated in FIGS. 11A, 11E and 11H or separated from one another asillustrated in FIGS. 11B-11D, 11F and FIG. A single chamber 54 can bedefined between the ultrasound transducers 20, 20A and the externalsurface 46 of the elongated body 14 as illustrated in FIGS. 11C, 11F and11G or a different chamber can be defined between each of the ultrasoundtransducers 20, 20A and the external surface 46. Although the ultrasoundtransducers 20, 20A in FIGS. 11A-11C are illustrated as having the samelongitudinal length, the longitudinal length may be different.

[0170] In FIGS. 11A-11H, the different temperature sensors can bepositioned adjacent to different ultrasound transducers 20, 20A. As aresult, the temperature adjacent to different ultrasound transducers 20,20A can be detected and the level of ultrasound energy produced by eachultrasound transducer adjusted in response to the detected temperature.

[0171] When the ultrasound assembly includes a second transducer 20A,the transducers 20, 20A may be constructed from the same or differentmaterials. Both transducers 20, 20A may be configured to radiateultrasound energy in the same direction. Further, one transducer may beconfigured to transmit ultrasound energy in a radial direction and theother in a longitudinal direction in order to increase the angularspectrum over which ultrasound energy can be simultaneously transmitted.The ultrasound transducers can be configured to transmit ultrasoundenergy having the same or different characteristics. The transmission ofultrasound energy with different characteristics allows the sameultrasound assemblies to be used to perform different functions. Forinstance, one ultrasound transducer can transmit a frequency which isappropriate for activating a light activated drug while the secondultrasound transducer transmits a frequency appropriate for enhancingpenetration of a therapeutic agent into the treatment site. Thetransducers can be operated independently or simultaneously. When thetransducers are operated simultaneously, the ultrasound assemblyproduces a waveform which is more complex than a single ultrasoundtransducer. More complex waveforms can provide advantages such as moreefficient rupture of microbubbles. It is also contemplated that theultrasound assembly can include three or more ultrasound transducersarranged similar to the transducers illustrated in FIGS. 11A-11H.

[0172] The ultrasound assembly 12 can be a separate module 58 asillustrated in FIGS. 12A-12B. In FIG. 12A, the catheter 10 includes afirst catheter component 60 a second catheter component 62 and anultrasound assembly module 58. The first and second catheter components60, 62 include component ends 64 which are complementary to theultrasound assembly module ends 66. The component ends 64 can be coupledwith the ultrasound assembly module ends 66 as illustrated in FIG. 12B.Suitable means for coupling the component ends 64 and the ultrasoundassembly module ends 66 include, but are not limited to, adhesive,mechanical and thermal methods. The ultrasound assembly 12 can beintegral with the catheter 10 as illustrated in FIG. 12C. Further, theouter coating 18 can have a diameter which is larger than the diameterof the elongated body 14 as illustrated in FIG. 10A or can be flush withthe external surface 46 of the elongated body 14 as illustrated in FIGS.12A-12C.

[0173] The ultrasound assembly 12 can be electrically coupled to produceradial vibrations of the ultrasound transducer 20 as illustrated inFIGS. 13A-13B. A first line 68 is coupled with an outer surface 70 ofthe ultrasound transducer 20 while a second line 72 is coupled with aninner surface 74 of the ultrasound transducer 20. The first and secondlines 68, 72 can pass proximally through the utility lumen 16 asillustrated in FIG. 13A. Alternatively, the first and second lines 68,72 can pass proximally through line lumens 76 within the catheter 10 asillustrated in FIG. 13B. Suitable lines for the ultrasound transducer 20include, but are not limited to, copper, gold and aluminum. Suitablefrequencies for the ultrasound energy delivered by the ultrasoundtransducer 20 include, but are not limited to, 20 KHz to 2 MHz.

[0174] The ultrasound assembly 12 can be electrically coupled to producelongitudinal vibrations of the ultrasound transducer 20 as illustratedin FIGS. 13C-13D. A first line 68 is coupled with a first end 78 of theultrasound transducer 20 while a second line 72 is coupled with a secondend 80 of the ultrasound transducer 20. The distal portion 82 of thesecond line 72 can pass through the outer coating 18 as illustrated inFIG. 13C. Alternatively, the distal portion 82 of the second line 72 canpass through line lumens 76 in the catheter 10 as illustrated in FIG.13D. As discussed above, the first and second lines 68, 72 can passproximally through the utility lumen 16.

[0175] As discussed above, the catheter 10 can include a plurality ofultrasound assemblies. When the catheter 10 includes a plurality ofultrasound assemblies, each ultrasound transducer 20 can each beindividually powered. When the elongated body 14 includes N ultrasoundtransducers 20, the elongated body 14 must include 2N lines toindividually power N ultrasound transducers 20. The individualultrasound transducers 20 can also be electrically coupled in serial orin parallel as illustrated in FIGS. 14A-14B. These arrangements permitmaximum flexibility as they require only 2 lines. Each of the ultrasoundtransducers 20 receive power simultaneously whether the ultrasoundtransducers 20 are in series or in parallel. When the ultrasoundtransducers 20 are in series, less current is required to produce thesame power from each ultrasound transducer 20 than when the ultrasoundtransducers 20 are connected in parallel. The reduced current allowssmaller lines to be used to provide power to the ultrasound transducers20 and accordingly increases the flexibility of the elongated body 14.When the ultrasound transducers 20 are connected in parallel, anultrasound transducer 20 can break down and the remaining ultrasoundtransducers 20 will continue to operate.

[0176] As illustrated in FIG. 14C, a common line 84 can provide power toeach ultrasound transducer 20 while each ultrasound transducer 20 hasits own return line 86. A particular ultrasound transducer 20 can beindividually activated by closing a switch 88 to complete a circuitbetween the common line 84 and the particular ultrasound transducer's 20return line 86. Once a switch 88 corresponding to a particularultrasound transducer 20 has been closed, the amount of power suppliedto the ultrasound transducer 20 can be adjusted with the correspondingpotentiometer 90. Accordingly, an catheter 10 with N ultrasoundtransducers 20 requires only N+1 lines and still permits independentcontrol of the ultrasound transducers 20. This reduced number of linesincreases the flexibility of the catheter 10. To improve the flexibilityof the catheter 10, the individual return lines 86 can have diameterswhich are smaller than the common line 84 diameter. For instance, in anembodiment where N ultrasound transducers 20 will be poweredsimultaneously, the diameter of the individual return lines 86 can bethe square root of N times smaller than the diameter of the common line84.

[0177] As discussed above, the ultrasound assembly 12 can include atleast one temperature sensor 22. Suitable temperature sensors 22include, but are not limited to, thermistors, thermocouples, resistancetemperature detectors (RTD)s, and fiber optic temperature sensors 22which use thermalchromic liquid crystals. Suitable temperature sensorgeometries include, but are not limited to, a point, patch, stripe and aband encircling the ultrasound transducer 20.

[0178] When the ultrasound assembly 12 includes a plurality oftemperature sensors 22, the temperature sensors 22 can be electricallyconnected as illustrated in FIG. 15. Each temperature sensor 22 can becoupled with a common line 84 and then include its own return line 86.Accordingly, N+1 lines can be used to independently sense thetemperature at the temperature sensors 22 when N temperature sensors 22are employed. A suitable common line 84 can be constructed fromConstantine and suitable return lines 86 can be constructed from copper.The temperature at a particular temperature sensor 22 can be determinedby closing a switch 88 to complete a circuit between the thermocouple'sreturn line 86 and the common line 84. When the temperature sensors 22are thermocouples, the temperature can be calculated from the voltage inthe circuit. To improve the flexibility of the catheter 10, theindividual return lines 86 can have diameters which are smaller than thecommon line 84 diameter.

[0179] Each temperature sensor 22 can also be independently electricallycoupled. Employing N independently electrically coupled temperaturesensors 22 requires 2N lines to pass the length of the catheter 10.

[0180] The catheter 10 flexibility can also be improved by using fiberoptic based temperature sensors 22. The flexibility can be improvedbecause only N fiber optics need to be employed sense the temperature atN temperature sensors 22.

[0181] The catheter 10 can be coupled with a feedback control system asillustrated in FIG. 16. The temperature at each temperature sensor 22 ismonitored and the output power of an energy source adjusted accordingly.The physician can, if desired, override the closed or open loop system.

[0182] The feedback control system includes an energy source 92, powercircuits 94 and a power calculation device 96 coupled with eachultrasound transducer 20. A temperature measurement device 98 is coupledwith the temperature sensors 22 on the catheter 10. A processing unit100 is coupled with the power calculation device 96, the power circuits94 and a user interface and display 102.

[0183] In operation, the temperature at each temperature sensor 22 isdetermined at the temperature measurement device 98. The processing unit100 receives signals indicating the determined temperatures from thetemperature measurement device 98. The determined temperatures can thenbe displayed to the user at the user interface and display 102.

[0184] The processing unit 100 includes logic for generating atemperature control signal. The temperature control signal isproportional to the difference between the measured temperature and adesired temperature. The desired temperature can be determined by theuser. The user can set the predetermined temperature at the userinterface and display 102.

[0185] The temperature control signal is received by the power circuits94. The power circuits 94 adjust the power level of the energy suppliedto the ultrasound transducers 20 from the energy source 92. Forinstance, when the temperature control signal is above a particularlevel, the power supplied to a particular ultrasound transducer 20 isreduced in proportion to the magnitude of the temperature controlsignal. Similarly, when the temperature control signal is below aparticular level, the power supplied to a particular ultrasoundtransducer 20 is increased in proportion to the magnitude of thetemperature control signal. After each power adjustment, the processingunit 100 monitors the temperature sensors 22 and produces anothertemperature control signal which is received by the power circuits 94.

[0186] The processing unit 100 can also include safety control logic.The safety control logic detects when the temperature at a temperaturesensor 22 has exceeded a safety threshold. The processing unit 100 canthen provide a temperature control signal which causes the powercircuits 94 to stop the delivery of energy from the energy source 92 tothe ultrasound transducers 20.

[0187] The processing unit 100 also receives a power signal from thepower calculation device 96. The power signal can be used to determinethe power being received by each ultrasound transducer 20. Thedetermined power can then be displayed to the user on the user interfaceand display 102.

[0188] The feedback control system can maintain the tissue adjacent tothe ultrasound transducers 20 within a desired temperature range for aselected period of time. As described above, the ultrasound transducers20 can be electrically connected so each ultrasound transducer 20 cangenerate an independent output. The output maintains a selected energyat each ultrasound transducer 20 for a selected length of time.

[0189] The processing unit 100 can be a digital or analog controller, ora computer with software. When the processing unit 100 is a computer itcan include a CPU coupled through a system bus. The user interface anddisplay 102 can be a mouse, keyboard, a disk drive, or othernon-volatile memory systems, a display monitor, and other peripherals,as are known in the art. Also coupled to the bus is a program memory anda data memory.

[0190] In lieu of the series of power adjustments described above, aprofile of the power delivered to each ultrasound transducer 20 can beincorporated in the processing unit 100 and a preset amount of energy tobe delivered may also be profiled. The power delivered to eachultrasound transducer 20 can then be adjusted according to the profiles.

[0191] The above catheters are suitable for locally delivering a mediaincluding a light activated drug. Suitable light activated drugsinclude, but are not limited to, fluorescein, merocyanin. However,preferred light activated drugs include xanthene and its derivatives andthe photoreactive pyrrole-derived macrocycles and their derivatives dueto a reduced toxicity and an increased biological affinity. Suitablephotoreactive pyrrole-derived macrocycles include, but are not limitedto, naturally occurring or synthetic porphyrins, naturally occurring orsynthetic chlorins, naturally occurring or synthetic bacteriochlorins,synthetic isobateriochlorins, phthalocyanines, naphtalocyanines, andexpanded pyrrole-based macrocyclic systems such as porphycenes,sapphyrins, and texaphyrins. Examples of suitable pyrrole-basedmacrocyclic classes are illustrated in FIG. 17A-17N.

[0192] As described above, the derivative of the pyrrole-basedmacrocycle classes can be used. For the purposes of illustrating some ofthe derivatives a macrocycle class, FIG. 17B-2 illustrates a formula forthe derivatives of texaphyrin: where M is H, CH₃, a divalent metalcation selected from the group consisting of Ca(II), Mn(II), Co(II),Ni(II), Zn(II), Cd(II), Hg(II), Fe(II), Sm(II), and UO(II) or atrivalent metal cation selected from the group consisting of Mn(III),Co(III), Ni(III), Fe(III), Ho(III), Ce(III), Y(III), In(III), Pr(III),Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Er(III), Tm(III),Yb(III), Lu(III), La(III), and U(III). Preferred metals include Lu(III),Dy(III), Eu(III), or Gd(III). M may be H or CH₃ in a non-metalated formof texaphyrin. R₁, R₂, R₃, R₄, R₅ and R₆ can independently be hydrogen,hydroxyl, alkyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, saccharide,carboxyalkyl, carboxyamidealkyl, a site-directing molecule, or a linkerto a site-directing molecule where at least one of R₁, R₂, R₃, R₄, R₅and R₆ is hydroxyl, hydroxyalkoxy, saccharide, alkoxy, carboxyalkyl,carboxyamidealkyl, hydroxyalkyl, a site-directing molecule- or a coupleto a site-directing molecule; and N is an integer less than or equal to2.

[0193] A preferred paramagnetic metal complex is the Gd(II) complex of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-(2-methoxyethoxy)ethoxy]ethoxy-13,20,25,26,27-pentaazapentacyclo[20.2.1.1^(3,6).1^(8,11).0^(14,19)]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene (“GdT2BET”) and a preferreddiamagnetic metal complex is the Lu(III) complex of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1.1^(3,6).1^(8,11).0^(14,19)]heptacosa-1,3,5,7,9,11,(27),12,

[0194] 14 (19),15,17,20,22(25),23-tridecaene (“LuT2BET”).

[0195] R₁, R₂, R₃, R₄, R₅ and R₆ may also independently be amino,carboxy, carboxamide, ester, amide sulfonato, aminoalkyl,sulfonatoalkyl, amidealkyl, aryl, etheramide or equivalent formulaeconferring the desired properties. In a preferred embodiment, at leastone of R₁, R₂, R₃, R₄, R₅ and R₆ is a site-directing molecule or is acouple to a site-directing molecule. For bulky R groups on the benzenering portion of the molecule such as oligonucleotides, one skilled inthe art would realize that derivatization at one position on the benzenepotion is more preferred.

[0196] Hydroxyalkyl means alkyl groups having hydroxyl groups attached.Alkoxy means alkyl groups attached to an oxygen. Hydroxyalkoxy meansalkyl groups having ether or ester linkages, hydroxyl groups,substituted hydroxyl groups, carboxyl groups, substituted carboxylgroups or the like. Saccharide includes oxidized, reduced or substitutedsaccharide; hexoses such as D-glucose, D-mannose or D-galactose;pentoses such as D-ribose or D-arabinose; ketoses such as D-ribulose orD-fructose; disaccharides such as sucrose, lactose, or maltose;derivatives such as acetals, amines, and phosphorylated sugars;oligosacchrides, as well as open chain forms of various sugars, and thelike. Examples of amine-derivatized sugars are galactosamine,glucosamine, and sialic acid. Carboxyamidealkyl means alkyl groups withsecondary or tertiary amide linkages or the like. Carboxyalkyl meansalkyl groups having hydroxyl groups, carboxyl or amide substitutedethers, ester linkages, tertiary amide linkages removed from the etheror the like.

[0197] For the above-described texaphyrins, hydroxyalkoxy may be alkylhaving independently hydroxy substituents and ether branches or may beC_((n−x))H_(((2n+1)−2x)O_(x)O_(y) or OC_((n−x))H_(((2n+1)−2x))O_(x)O^(y)where n is a positive integer from 1 to 10, x is zero or a positiveinteger less than or equal to n, and y is zero or a positive integerless than or equal to ((2n+1)_(—)2×). The hydroxyalkoxy or saccharidemay be C_(n)H_(((2n+1)−q))O_(y)R^(a) _(q),OC_(n)H_(((2n+1)−q))O_(y)R^(a) _(q) or (CH₂)_(n)CO₂R^(a) where n is apositive integer from 1 to 10, y is zero or a positive integer less than((2n+1)−q), q is zero or a positive integer less than or equal to 2n+1,and R^(a) is independently H, alkyl, hydroxyalkyl, saccharide,C_((m−w))H_(((2m+1)−2w))O_(w)O_(z),O₂CC_((m−w))H_(((2m+1)−2w))O_(w)O_(z) orN(R)OCC_((m−w))H_(((2m+1)−2w))O_(w)O_(z). In this case, m is a positiveinteger from 1 to 10, w is zero or a positive integer less than or equalto m, z is zero or a positive integer less than or equal to ((2m+1)−2w), and R is H, alkyl, hydroxyalkyl, or C_(m)H_(((2m+1)−r)O)_(z)R^(b) _(r) where m is a positive integer from 1 to 10, z is zero ora positive integer less than ((2 m+1)−r), r is zero or a positiveinteger less than or equal to 2 m+1, and R^(b) is independently H,alkyl, hydroxyalkyl, or saccharide.

[0198] Carboxyamidealkyl may be alkyl having secondary or tertiary amidelinkages or (CH₂)_(n)CONHR^(a), O(CH₂)_(n)CONR^(a),(CH₂)_(n)CON(R^(a))2, or O(CH₂)_(n)CON(R^(a))2 where n is a positiveinteger from 1 to 10, and R is independently H, alkyl, hydroxyalkyl,saccharide, C_((m−w))H_(((2m+1)2w))O_(w)O_(z),O₂CC_((m−w))H_(((2m+1)2w))O_(w)O_(z), N(R)OCC_((m−w))H_(((2m+1)2w)O)_(w)O_(z), or a site-directing molecule. In this case, m is a positiveinteger from 1 to 10, w is zero or a positive integer less than or equalto ((2M+1)_(—)2w), and R is H, alkyl, hydroxyalkyl, orC_(m)H_(((2m+1)r))O_(z)R^(b) _(r). In this case, m is a positive integerfrom 1 to 10, w is zero or a positive integer less than or equal to m, zis zero or a positive integer less than or equal to ((2M+1)_r), r iszero or a positive integer less than or equal to 2 m+1, and R^(b) isindependently H, alkyl, hydroxyalkyl, or saccharide. In a preferredembodiment, R^(a) is an oligonucleotide.

[0199] Carboxyalkyl may be alkyl having a carboxyl substituted ether, anamide substituted ether or a tertiary amide removed from an ether orC_(n)H_(((2n+1)−q))O_(y)R^(c) _(q) or OC_(n)H_(((2n+1)−q))O_(y)R^(c)_(q) where n is a positive integer from 1 to 10; y is zero or a positiveinteger less than ((2n+1)−q), q is zero or a positive integer less thanor equal to 2n+1, and R^(c) is (CH₂)_(n)CO₂R^(d), (CH₂)_(n)COHR^(d),(CH₂)_(n)CON(R^(d))₂ or a site-directing molecule. In this case, n is apositive integer from 1 to 10, R^(d) is independently H, alkyl,hydroxyalkyl, saccharide, C_((m−w))H_(((2m+1)−2w))O_(w)O_(z),O₂CC_((m−w))H_(((2m+1)−2w))O_(w)O_(z) or N(R)OCC_((m−w))H_(((2m+1)−2w)O)_(w)O_(z). In this case, m is a positive integer from 1 to 10, z is zeroor a positive integer less than ((2 m+1)_(—)2w), and R is H, alkyl,hydroxyalkyl, or C_(m)H_(((2m+1)r))O_(z)R^(b) _(r). In this case, m is apositive integer from 1 to 10, z is zero or a positive integer less than((2 m+1)−r), r is zero or a positive integer less than or equal to 2M+1,and R^(b) is independently H, alkyl, hydroxyalkyl, or saccharide. In apreferred embodiment, R^(c) is an oligonucleotide.

[0200] Exemplary texaphyrins are listed in Table 1. TABLE 1Representative Substitutes for Texaphyrin Macrocycles TXP R₁ R₂ R₃ R₄ R₅R₆ A1 CH₂(CH₂)₂OH CH₂CH₃ CH₂CH₃ CH₃ O(CH₂)₃OH O(CH2)3OH A2 ″ ″ ″ ″O(CH₂CH₂O)₃CH₃ O(CH₂CH₂O)₃CH₃ A3 ″ ″ ″ ″ O(CH₂)_(n)CON-linker-site- ″directing molecule, n- = 1-7 A4 ″ ″ ″ ″ O(CH₂)_(n)CON linker- Hsite-directing molecule A5 ″ ″ ″ ″ OCH₂CO-hormone ″ A6 ″ ″ ″ ″O(CH₂CH₂O)₃CH₃ ″ A7 ″ ″ ″ ″ OCH₂CON-linker-site- O(CH₂CH₂O)₃CH₃directing molecule A8 ″ ″ ″ ″ OCH₂CO-hormone ″ A9 ″ ″ ″ ″O(CH₂CH₂O)₁₂₀CH₃ O(CH₂CH₂O)₃ CH₂—CH₂—N- imidazole A10 ″ ″ ″ ″ saccharideH A11 ″ ″ ″ ″ OCH₂CON—(CH₂CH₂OH)₂ ″ A12 ″ ″ ″ ″ CH₂CON(CH₃)CH₂— ″(CHOH)₄CH₂OH A13 ″ COOH COOH ″ CH₂CON(CH₃)CH₂— ″ (CHOH)₄CH₂OH A14 ″COOCH₂CH₃ COOCH₂CH₃ ″ CH₂CON(CH₃)CH₂— ″ (CHOH)₄CH₂OH A15Ch₂CH₂CON(CH₂CH₂OH)₂ CH₂CH₂ CH₂CH₃ ″ CH₂CON(CH₃)CH₂— ″ (CHOH)₄CH₂OH A16CH₂CH₂ON(CH₃)CH2— ″ ″ ″ OCH₃ OCH₃ (CHOH)₄CH₂OH A17 CH₂(CH₂)₂OH ″ ″ ″O(CH₂)_(n)COOH, n = 1-7 H A18 ″ ″ ″ ″ (CH₂)_(n)—CON-linker-site- ″directing molecule, n = 1-7 A19 ″ ″ ″ ″ YCOCH₂-linker-site-directing ″molecule - Y = NH, O A20 CH₂CH₃ CH₃ CH₂CH₂COOH ″ O(CH₂)₂CH₂OHO(CH₂)₂CH₂OH A21 ″ ″ CH₂CH₂CON- ″ ″ ″ oligo A22 CH₂(CH₂)₂OH CH₂CH₃CH₂CH₃ ″ O(CH₂)₃CO-histamine H

[0201] Preferred pyrrole-based macrocycles include, but are not limitedto the hydro-monobenzoporphyrins (the so-called “fi porphyrine” or “Gp”compounds) disclosed in U.S. Pat. Nos. 4,920,143 and 4,883,790 which areincorporated herein by reference. Typically, these compounds are poorlywater-soluble (less than 1 mg/ml) or water-insoluble. Gp is preferablyselected from the group consisting of those compounds having one of theformulae A-F set forth in FIGS. 18A-F, mixtures thereof, and themetalated and labeled forms thereof.

[0202] In FIGS. 18A-F, R¹ and R² can be independently selected from thegroup consisting of carbalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl(6-10C), sulfonyl, aryl (6-10C), cyano, and —CONR⁵CO— wherein R⁵ is aryl(6-10C) or alkyl (1-6C). Preferably, however, each of R¹ and R² iscarbalkoxy (2-6C). R³ can be independently carboxyalkyl (2-6C) or asalt, amide, ester or acylhydrazone thereof, or is alkyl (1-6C).Preferably R³ is —CH²CH²COOH or a salt, amide, ester or acylhydrazonethereof.

[0203] R⁴ is —CHCH₂; —CHOR^(4′) wherein R^(4′) is H or alkyl (1-6C),optionally substituted with a hydrophilic substituent; —CHO; —COOR^(4′);CH(OR⁴)CH₃; CH(OR^(4′)) CH₂OR^(4′); —CH(SR^(4′))CH₃; —CHNR^(4′) ₂)CH₃;—CH(CN)CH₃; —CH(COOR^(4′))CH₃; —CH(OOCR^(4′))CH₃; —CH(halo)CH₃;—CH(halo)CH₂(halo); an organic group of <12C resulting from direct orindirect derivatization of a vinyl group; or R⁴ consists of 1-3tetrapyrrole-type nuclei of the formula -L-P, wherein -L- is selectedfrom the group consisting of

[0204] and P is a second Gp, which is one of the formulae A-F (FIG. 18)but lacks R⁴, or another porphyrin group. When P is another porphyringroup, P preferably has the formula illustrated in FIG. 19: wherein eachR is independently H or lower alkyl (1-4C); two of the four bonds shownas unoccupied on adjacent rings are joined to R³; one of the remainingbonds shown as unoccupied is joined to R⁴; and the other is joined to L;with the proviso that, if R⁴ is —CHCH₂, both R³ groups cannot becarbalkoxyethyl. The preparation and use of such compounds is disclosedin U.S. Pat. Nos. 4,920,143 and 4,883,790, which are hereby incorporatedby reference.

[0205] Even more preferred for including in liposomes are lightactivated drugs that are designated as benzoporphyrin derivatives(“BPD's”). BPD's are hydrolyzed forms, or partially hydrolyzed forms, ofthe rearranged products of formula A-C or formula A-D, where one or bothof the protected carboxyl groups of R³ are hydrolyzed. Particularlypreferred is the compound referred to as BPD-MA in FIGS. 20A-D, whichhas two equally active regioisomers.

[0206] As described above, activating a light activated drug included ina microbubble can enhance rupture of the microbubble. Preferred lightactivated drugs for including in a microbubble to enhance rupture of themicrobubble include Hematporphyrin, Rose Bengal, Eosin Y, Erythrocin,Rhodamine B, and PHOTOFRIN. The formulae for these preferred lightactivated drugs are illustrated in FIGS. 21A-B where Rose Bengal, EosinY, Erythrocin and Rhodamine B are xanthene derivatives.

[0207] The present invention has a first characteristic in that byadopting ultrasound-sensitive substance-containing drug carriers thatcan carry a therapeutic drug and transport it to a target location, whensaid drug carriers carrying drugs are irradiated with ultrasound at thetarget location, a chemical change or a physical change occurs in saidultrasound-sensitive substance so that said drug carriers rupture torelease the drugs.

[0208] In comparison to the method in which the capsule shells aresimply ruptured by the action of vibration due to ultrasound in order torelease the drugs inside, the rupture of capsules according to thepresent invention is not as greatly affected by the ultrasoundirradiation conditions, so it is possible to adopt a relatively widerange of ultrasound energies, namely 0.1-1000 watts/cm². Therefore,there is no setting of extremely difficult ultrasound irradiationconditions, as the drug carriers can be ruptured and the drugs releasedefficiently at the target locations within the body even with ultrasoundof frequencies other than the resonance frequency.

[0209] In addition, the present invention has a second characteristic inthat hollow areas in the drug carriers are formed by shell walls thathave a prescribed thickness, and said shell walls contain or are coatedor covered with an ultrasound-sensitive substance. To wit, the drugcarriers are designed so that while they are sufficiently resistant topressure and other types of mechanical energy, their structure is suchthat they are ruptured easily by the chemical changes or physicalchanges in said ultrasound-sensitive substance.

[0210] Specifically, the drug carrier takes the form of a capsule or thelike, and the shell walls that make up the capsule contain or are coatedor covered with an ultrasound-sensitive substance in a laminar manner orin lumps. In the case that the aforementioned ultrasound-sensitivesubstance is disposed in a laminar manner, then the effects ofmodification of said ultrasound-sensitive substance may extend over theentire aforementioned shell walls. On the other hand, in the case thatthe aforementioned ultrasound-sensitive substance is disposed in lumps,the said ultrasound-sensitive substance is present locally in said shellwalls, making it possible for said shell walls to be ruptured reliably.Note that a uniform mixture of the ultrasound-sensitive substance in asubstrate to be described later may be used as the substance that makesup the drug carrier itself. In this case, not only will the shell wallsbe ruptured uniformly, but the fabrication of the drug carrier will besimplified.

[0211] Moreover, the present invention has a third characteristic inthat a drug is carried in the hollow areas formed by the shell walls,but a prescribed amount of gas may also be present in said hollow areastogether with said drug. As described later, the type and amount of saidgas is arbitrary, but this should be preferably set in the range of0.01-50% of the volume of said hollow areas. There follows a descriptionof the composition and structure of the drug carriers.

[0212] Here, a “drug carrier” is defined to be a carrier that can carrya therapeutic drug and transport it to a target location. While its formis not particularly limited, in consideration of the ease ofmanufacture, manufacturing costs and other considerations, a capsuleform having a hollow area which is isolated from the outside by a shellwall is preferable.

[0213] The size of the drug carrier is normally set appropriately in therange of 0.01-100 μm. If less than 0.01 μm, then it will be excretedoutside of the body and be inadequately effective. If greater than 100μm, then there is a risk of interfering with the flow of blood withinthe blood vessels.

[0214] The substrate material that is used for the drug carrier may beone of various biocompatible polymers, albumins, liposomes, sugars orother substances.

[0215] In addition, by using a drug carrier that has been modified tothe prodrug form, the selective movement to the locations of taggedtissue can be improved, aqueous solubility can be increased, absorptioncan be promoted and side effects can be lessened. In this case, theprodrug reverts to the original drug carrier in an enzymatic ornon-enzymatic manner in the body after it reaches the target of theaforementioned modification, so it is possible for the sensitivity toultrasound to be restored. A drug carrier that has been modified to theprodrug form is included in the scope of the present invention. Notethat it is also appropriate for the drug carried by the drug carrier tobe modified to the prodrug form.

[0216] An ultrasound-sensitive substance is defined to be a substancethat is activated by ultrasound of a prescribed frequency and intensityby means of a mechanism to be described later, and which causes chemicalchanges to occur in itself or another substance, or changes its ownstructure or causes some sort of changes to occur. Examples ofultrasound-sensitive substances include fluorescein, merocyanin, and thelike, but from the standpoint of toxicity and affinity with respect tothe body, porphyrin derivatives or xanthene derivatives are preferable.Specific examples of the aforementioned porphyrin derivatives orxanthene derivatives are discussed in more detail later.

[0217] Moreover, with regard to the structure of the drug carrier, asshown in FIG. 22, lumps of the drug 108 and ultrasound-sensitivesubstance 110 may be distributed at appropriate intervals within a drugcarrier 106 of indefinite shape, but it is preferable to shape the drugcarrier itself so that it has shell walls having a prescribed thicknessto form hollow areas therein, and have said shell walls contain or becovered with the aforementioned ultrasound-sensitive substance to permitsaid drug carrier to be ruptured efficiently.

[0218] If the aforementioned hollow areas are formed on the interior ofthe drug carrier, there are no particular limits to the numbers thereof,as there may be one or more. In addition, there are no particular limitsto their shapes or locations, but they are preferably formed in thesurface layers of the drug carrier in order for the drug release to beperformed well. Note that making the shape of the drug carrier itself asthat of a capsule consisting of layers of shell walls that have theaforementioned prescribed thickness is also contained in the scope ofthe present invention.

[0219] Here, the thickness of the aforementioned shell walls is normallydetermined within the range of 0.001-50 μm. If the thickness of theshell walls is less than 0.001 μm, then the shell walls become easilyruptured by shock and so there is a risk of the aforementioned shellwalls being ruptured and drug inside leaking out before the targetlocation within the body is reached. On the other hand, if 50 μm orthicker, rupturing the aforementioned shell walls becomes difficult evenwith the action of the ultrasound-sensitive substance, and even if aportion of the shell walls is ruptured, there is a risk thatunrupturable shell wall portions remain, blocking the release of drugs.

[0220] At the time that an ultrasound-sensitive substance is containedin the drug carrier, if said drug carrier is equipped with a shell wallstructure, it is preferable that the aforementioned ultrasound-sensitivesubstance have said shell walls contain or be covered with theaforementioned ultrasound-sensitive substance.

[0221] Specifically, the outside surface of shell wall 112 may becovered with a layer of the ultrasound-sensitive substance 110 as shownin FIG. 23(a), or the inside surface of the shell wall 112 may be coatedwith a layer of the substance as shown in FIG. 23(b), or a layer of thesubstance may be present entirely within the interior of shell wall 112in a laminar manner as shown in FIG. 23(c). Note that the layer of theultrasound-sensitive substance 110 need not have a continuous layerstructure as shown in FIG. 23, and moreover the layer need not beconcentric with the shell wall 112 as shown in FIG. 23.

[0222] In addition, the shell wall 112 may contain or be covered withthe ultrasound-sensitive substance 110 in a manner such that it isdistributed in lumps. To wit, as shown in FIGS. 24(a) and 24(b), lumpsof the ultrasound-sensitive substance 110 may be adhered to the outsidesurface of inside surface of said shell wall 112, or as shown in FIG.24(c), lumps may be distributed appropriately within the interior ofsaid shell wall 112. In this case, portions of the individual lumps mayprotrude from the shell wall 112 or they need not protrude.

[0223] Moreover, as shown in FIG. 24(d), the lumps of theultrasound-sensitive substance 110 may penetrate through the shell wall112 in a structure such that portions of the lumps are exposed to theoutside and the interior space.

[0224] In the cases shown in FIG. 24, lumps of the ultrasound-sensitivesubstance 110 may be distributed uniformly within the aforementionedshell wall 112, or the lumps may be distributed non-uniformly. Inaddition, there are no particular limitations to the shapes of theindividual lumps, as shapes other than spherical are also possible.

[0225] Note that in FIGS. 23 and 24, the numeral 114 indicates thehollow area formed by the shell wall 112 so that various types of drugsare carried in this hollow area.

[0226] The method of using the drug carrier described above will now beexplained with reference to FIG. 25.

[0227] A drug of a prescribed type carried in the abovementioned drugcarrier is administered orally or administered hypodermically using asyringe or other drug administration apparatus, or using a specialmethod, injected into the body by means of the iontophoresis methodusing ionized drug carriers.

[0228] Moreover, in the case that the use of a catheter or endoscope ispossible, a therapeutic ultrasound generator is attached to the tip of acatheter or endoscope and introduced into the interior of the body ofthe patient and allowed to reach the affected area.

[0229] FIGS. 25(a) and 25(b) are both sectional drawings showing themethod of attaching the ultrasound generator used in the working of thepresent invention, where FIG. 25(a) shows the structure in the case inwhich the ultrasound generator is attached to the tip of an endoscope,while FIG. 25(b) shows the structure in the case in which it is attachedto the tip of a catheter.

[0230] In the manner shown in FIG. 25(a), a miniature center tube 118containing an optical fiber (not shown) and wiring used to operateultrasonic oscillators (to be described later) is laid in the interiorof a fine tube 116 constituting the endoscope. A cylindrical firstultrasonic oscillator 124 and second ultrasonic oscillator 126 aredisposed concentrically on the tip of the fine tube 116. Examples of theultrasonic oscillators include one made by mounting electrodes on eitherside of a piezoelectric element, and in this case, ultrasound isgenerated by applying an electrical signal with an ultrasonic frequencybetween said electrodes. Numeral 122 is a core used to transmit an imageof the outside to the optical fiber (not shown) embedded in theaforementioned hollow area. In addition, the gap between the fine tube116 and the miniature center tube 118 becomes the drug delivery path 120connected to penetration holes 128 opened at appropriate intervals inthe circumferential surface of the tip side of the fine tube 116.

[0231] Moreover, the respective frequency characteristics of theaforementioned first ultrasonic oscillator 124 and second ultrasonicoscillator 126 are different, so by controlling the operation of both ofthese, a mixture of two different frequencies can be generated in adirection (the direction indicated by arrows) perpendicular to the axialdirection of the endoscope. Radiating a combination of differentultrasound frequencies in this manner is done because a more complexultrasound waveform improves the efficiency of rupturing the drugcarriers. Note that the first and second ultrasonic oscillators havingthe structure described above may also be mounted on the tip of acatheter.

[0232] On the other hand, the first ultrasonic oscillator 124 and secondultrasonic oscillator 126 with different frequency characteristics mayboth have a solid cylindrical shape and be laminated in the direction ofthe axis of the catheter in the manner shown in FIG. 25(b).

[0233] Therefore, by controlling the operation of both of these,ultrasound having two different frequencies can be generated in theaxial direction of the endoscope (the direction indicated by arrows).

[0234] Note that the first and second ultrasonic oscillators having thestructure described above may also be mounted on the tip of anendoscope.

[0235] Now, after confirming that the tip of the endoscope or catheterhas reached the affected tissue, the drug carriers are released towardthe affected part-along the drug delivery path 120 through thepenetration holes 128. At the same time or after a prescribed amount oftime has elapsed, the first and second ultrasonic oscillators areoperated thereby irradiating the affected area with ultrasound ofdifferent frequencies, thus rupturing the drug carriers present in theaffected tissue. Therefore, the drug within the drug carrier isadministered over a range limited to the vicinity of the location of theaffected tissue.

[0236] Considering the location of the affected area relative to theaforementioned endoscope or catheter, it is preferable to select theequipment such that suitable irradiation of ultrasound is obtained. Inaddition, the diameter of the aforementioned endoscope or catheter maybe selected appropriately as one in the range from 1 mm to 5 cm.

[0237] Note that three or more types of the aforementioned ultrasonicoscillators may also be mounted. In this case, an even more complexultrasound waveform can be generated, so the efficiency of rupturing thedrug carriers can be increased even further. A single ultrasonicoscillator may be used, for example, if the drug carriers have a shapethat is easily ruptured.

[0238] On the other hand, in the event that a catheter or endoscopecannot be used, as shown in FIG. 26, a first ultrasonic oscillator 124and a second ultrasonic oscillator 126 having the same characteristicsas those described above may be laminated and disposed in a hollow areaof a base 130 consisting of flexible synthetic resin or the like. One ora plurality of such therapeutic ultrasound generators are placed uponthe skin in areas corresponding to the affected areas and irradiateultrasound toward the affected areas (in the direction indicated byarrows). As shown in the figure; it is preferable to provide a pluralityof laminates of ultrasonic oscillators in the base 130. The base 130 canflex to match the shape of the patient's body so the ultrasound can beconcentrated in the affected area. The diameter of the aforementionedfirst and second ultrasonic oscillators is normally set appropriately inthe range of 5-10 cm. Note that it is preferable for the aforementionedfirst and second ultrasonic oscillators to be made of flexibleoscillating material such as a fluorine compound in order to maintainthe overall flexibility of the apparatus and keep its tight fit to theskin or the like.

[0239] Moreover, the ultrasound energy concentrated in the affected arearuptures the drug carriers present there and releases the drug into theinterior of the affected area.

[0240] In this manner, according to the present invention, by includingan ultrasound-sensitive substance in the drug carrier, a drug carrierthat is difficult to rupture with ultrasound only can be easily rupturedand the release of the drug into a desired location in the body can becontrolled.

[0241] Note that in the case that substances such as antibodies thatselectively bind to cancerous cells, thrombi, organs, blood vessels,hardened arteries or the like are included in the substrate material ofthe drug carrier, the drug carriers can be concentrated in the tissue ofthe affected area, so it is possible to use ultrasound to rupture thedrug carriers concentrated in said tissue of the affected area toadminister the drug locally in high concentration.

[0242] Here follows a description of the conditions for the ultrasoundused in the present invention.

[0243] The power of ultrasound irradiated in order to rupture the drugcarriers is set appropriately in the range of 0.1-1000 watts/cm². If thepower of ultrasound is below 0.1 W/cm², there is insufficient energy toactivate the ultrasound-sensitive substance, and above 1000 W/cm²,excessive amounts of heat are generated, causing damage to the body.

[0244] In addition, the frequency of the ultrasound is set appropriatelyin the range 10 kHz-100 MHz, but the range 20 kHz-10 MHz is particularlypreferable. With ultrasound in this frequency band, a relatively lowenergy is able to cause the cavitation to be described later andefficiently rupture the drug carriers.

[0245] Thus, by combining multiple frequencies as described above, it ispossible to activate the ultrasound-sensitive substance and efficientlyrupture the drug carriers. For example, during irradiation withultrasound of a fixed frequency, intermittently switching between thisand a different frequency can be performed intentionally to enhance orsuppress the generation of the cavitation to be described later and thusrupture or disintegrate the drug carriers.

[0246] Thus, by combining multiple frequencies as described above, it ispossible to activate the ultrasound-sensitive substance and efficientlyrupture the drug carriers. For example, during irradiation withultrasound of a fixed frequency, intermittently switching between thisand a different frequency can be performed intentionally to enhance orsuppress the generation of the cavitation to be described later and thusrupture or disintegrate the drug carriers.

[0247] To present an actual example, while the affected area iscontinuously irradiated with ultrasound at 100 kHz, switching to afrequency of 270 kHz in short (0.001-10 sec) pulses have even bettereffectiveness of rupturing. The same effect is expected if the frequencyof the ultrasound is continuously varied within a fixed range. Thisphenomenon is thought to increase the rupture forces by temporarilyhalting the resonance movement of the drug carrier.

[0248] Here follows an explanation of the mechanism of rupturing of thedrug carriers.

[0249] Typically, when ultrasonic energy above a certain value isapplied to a liquid, the phenomenon called cavitation occurs, whereinminute air bubbles form. The mechanism of generation of cavitation isdescribed in Robert E. Apfel: “Sonic effervescence: tutorial on acousticcavitation,” Journal of Acoustic Society of America 101 (3): 1227-1237,March 1997, Atchley A. Crum L.: “Ultrasound—Its chemical, physical andbiological effects: Acoustic cavitation and bubble dynamics,” Ed.Suslick, K. pp. 1-64, 1988 VCH Publishers, New York, and this isdescribed briefly below.

[0250] Cavitation is a phenomenon in which gas dissolved in an aqueoussolution forms bubbles under certain types of acoustic vibration, or inwhich extremely small bubbles already present oscillate or repeatedlyenlarge and contract, becoming bubbles. Then, when the size of thesecavitation bubbles reaches a size that cannot be maintained, theycollapse and this collapse occurs suddenly so it is known that varioustypes of energy occur locally at this time.

[0251] To wit, at the time of the aforementioned cavitation collapse,hot spots of 6000-7000 degrees are formed at the center, so in additionto vibration and other types of mechanical energy, visible light,ultraviolet light and other types of electromagnetic radiation, heat,plasma, magnetic fields, shock waves, free radicals, heat and otherforms of energy are thought to be generated locally.

[0252] The ultrasound-sensitive substance according to the presentinvention is activated by the aforementioned various forms of energygenerated at the time of cavitation collapse, and also chemical changesare thought to occur along with structural changes.

[0253] For example, one of the ultrasound-sensitive substances accordingto the present invention, Rose Bengal, is excited and activated by lightof wavelength 530 nm or ultraviolet light. Therefore, the activation ofRose Bengal is thought to be caused by the ultraviolet light generatedat the time of cavitation collapse.

[0254] The threshold value of ultrasonic energy for cavitation to occuris known to become lower in a liquid in which an ultrasound-sensitivesubstance is present. Therefore, the ultrasound-sensitive substancecontained in the drug carrier exhibits the effects of selectivelyinducing the occurrence of cavitation in the vicinity of the drugcarrier, and also itself being activated by the energy generated by thecollapse of cavitation to rupture the drug carrier.

[0255] On the other hand, it is known that the threshold of ultrasonicenergy in order to generate cavitation also becomes lower if minutebubbles are present in the liquid. Therefore, cavitation can beeffectively caused to occur during ultrasound irradiation by causing aprescribed amount of gas to be present within the drug carrier, and theenergy generated at the time of this cavitation collapse can be usedeffectively to rupture the drug carrier. The type and amount of said gasis arbitrary, but the amount is preferably set in the range of 0.01-50%of the volume of the hollow area, then the generation of cavitationcannot be effectively induced, and if the amount of same gas exceeds 50%of the volume of said hollow area, the drug carrier may not havesufficient strength to reach the target location, and the amount of drugtransported is limited.

[0256] Note that the visible light, ultraviolet light and other types ofelectromagnetic radiation, heat, plasma, magnetic fields, shock waves,free radicals, heat and other forms of energy generated at the time ofcollapse of cavitation can be used directly in the treatment of affectedtissue.

[0257] For example, by generating ultrasound in the vicinity of theaffected tissue, ultraviolet light that is normally absorbed by the skinand does not reach the interior of the body can be generated in thevicinity of affected tissue within the body by means of the collapse ofcavitation derived from ultrasound, so it is possible to treat affectedareas by its disinfectant action.

[0258] To wit, cavitation can be induced within the body by means ofultrasound and the energy generated at the time of the collapse can beused in the treatment of affected areas. By means of this method,ultraviolet light and other forms of energy can be generated at will atany location within the body to perform treatment of affected areas, sothere would be no need to consider the side effects specific to drugtherapy.

[0259] The present invention can be worked in the manner describedbelow:

[0260] Cancer Therapy

[0261] The anti-cancer drug cisplatin is enclosed in a polymer capsule,and then the outside surface of this capsule is coated with anultrasound-sensitive substance and the resulting capsule is injectedarterially. Since the cisplatin is covered with a polymer, thesecapsules have no toxicity (side effects) even when injected arterially,as they merely flow along in the blood.

[0262] However, when these capsules flow along inside the blood vesselsinside cancerous tissue and are irradiated with ultrasound, theultrasound-sensitive substance on the surface of the capsule isactivated so the capsule enclosing the cisplatin disintegrates and thecisplatin is released inside this tissue in high concentrations.Therefore, the administration of anti-cancer drugs in highconcentrations is possible in limited areas where cancer is located, yetnormal cellular tissue can be spared the strong toxicity of cisplatin.

[0263] This method is particularly effective in cancer of the liver,brain tumors and other diseases in which large numbers of blood vesselsare present. The method of irradiation with ultrasound may beirradiation of the tumor portions from the surface of the skin, orirradiation of the cancerous tissue with ultrasound directly during alaparotomy.

[0264] In addition, it is also possible to attach ultrasound generatorsto an endoscope and irradiate the interior of the body with ultrasound,and in this case, the interior of the stomach and colon cancers and thelike can be directly irradiated with ultrasound from bodily cavities inthe inside of the colon.

[0265] The route of administration of these capsules may be arterialinjection or absorption from the intestines after oral administration,direct injection into the affected area via lymphatic vessels and otherroutes. The appropriate route can be selected depending on the case.

[0266] However, if Photofrin® is used as the aforementionedultrasound-sensitive substance, since Photofrin® itself has an affinityfor cancer cells, the aforementioned cisplatin-containing capsulesconcentrate in the cancerous tissue and accumulate in highconcentration.

[0267] Moreover, cisplatin can be administered in cancerous tissue ateven higher concentrations by irradiating with ultrasound in this state.Note that when Photofrin®is activated by ultrasound, it also has thecharacter of causing a cell-killing action, so in this case, theanti-cancer action is augmented in a synergistic manner with cisplatin.

[0268] In addition, in the case of cancer that has metastasized withinthe abdomen or bladder cancer, a conceivable therapy method would be toinject capsules containing anticancer drugs directly into the interiorof the abdomen and then irradiate the entire abdomen with ultrasoundfrom the surface of the skin. In the case of bladder cancer, theinterior of the bladder can be filled with these capsules from theurethra and the bladder cancer can be treated by irradiating the lowerabdomen with ultrasound from the surface of the skin. In these cases,there is a an added advantage in that the state of release of drugs canbe observed using diagnostic ultrasound equipment during irradiationwith therapeutic ultrasound.

[0269] Thrombolytic Therapy

[0270] Thrombolytic agents are used as drugs for the treatment ofmyocardial infarction and cerebral infarction. However, if large dosesof drugs are administered in order to dissolve the thrombi as fast aspossible, there is a risk of the blood conversely becoming less easilyclotted, resulting in excessive hemorrhaging.

[0271] To solve this problem, urokinase or another thrombolytic agent isenclosed in capsules made of biocompatible polymer or albumin whichcontains an ultrasound-sensitive substance and the resulting capsulesare injected into a blood vessel. Since these capsules are not rupturedin the normal state, they do not induce thrombolytic action within theblood vessels.

[0272] In the case of a myocardial infarction, for example, when thesecapsules reach the peripheral blood vessels of the coronary artery orother locations where thrombi are present, an apparatus such as thatdescribed above can be used to irradiate these locations with ultrasoundfrom outside the body or inside the body, thus rupturing the capsulesand releasing the drug locally in high concentrations.

[0273] Specifically, as shown in FIG. 27, the catheter or endoscope 132equipped with ultrasound generators as shown in FIG. 25 is inserted intothe interior of the blood vessel 134 up until near the thrombus 136 andthe thrombolysis agent-containing capsules 138 are released immediatelyupstream of the thrombus 136 while ultrasound is simultaneouslygenerated. These capsules are ruptured by the action of theultrasound-sensitive substance which his activated by ultrasound, so thethrombolysis agent inside is released to the location of the thrombus.

[0274] Note that the capsules that were not ruptured by ultrasound thefirst time are ruptured and used when they again flow back to theaffected areas. These capsules are not ruptured during the period beforethey again reach the affected area so they will not become the cause ofhemorrhaging.

[0275] In addition, if a substance or antibody with particular affinityto thrombi is attached to the outside surface of the capsules, thesecapsules will collect at a thrombus in high concentrations. If thelocation of the thrombus is irradiated with ultrasound in this state,these capsules can be ruptured to administer the thrombolysis agenteffectively in the vicinity of the thrombus. A substance that hasparticular affinity to thrombi has been reported by Lanza, et al.(Circulation, 1995, 92, Suppl. I: 1-260). By doing such, thrombolytictherapy can be performed efficiently without causing hemorrhaging orother side effects.

[0276] Blood Vessel Therapy

[0277] In the case that angiostenosis due to arteriosclerosis or thelike occurs, a surgical treatment in which a balloon catheter is used toenlarge the cavity within the blood vessel to reopen the flow of bloodhas become common in recent years. In addition, a procedure in which ametal stent is used to fix the cavity within the blood vessel in theenlarged state so that the narrowing of blood vessels does not recurafter the above surgery has also become common. However, in either case,the inside walls of the blood vessel are damaged to a certain extent. Inorder to restore the damaged blood vessel tissue to the original stateof the blood vessel, repair, including the grafting of blood vesseltissue is performed, but excessive repair in the repair process occursin more than 50% of cases, leading to recurrence of angiostenosis. Thisis a drawback of this therapy.

[0278] To solve this problem, as shown in FIG. 28, drug carriers 140that carry Photofrin® internally are mixed into the material for theballoon 146, and a balloon catheter with an ultrasound generator 132positioned in its center 142 is inserted into the blood vessel 144. Theballoon 146 is inflated at the target location and brought into closecontact with the blood vessel walls 144. When ultrasound is generated inthe direction perpendicular to the axis of the catheter, the drugcarriers 140 located in areas of the balloon 146 in contact with theblood vessel walls 144 are ruptured and the Photofrin® inside isinjected directly into the blood vessel walls 144.

[0279] When ultrasound is generated in the direction perpendicular tothe axis of the catheter, the drug carriers located in areas of theballoon in contact with the blood vessel walls are ruptured and thePhotofrin® inside is injected directly into the blood vessel walls. Whenactivated with ultrasound, Photofrin® has the character of blocking theblood vessel tissue repair process to a certain degree so it is possibleto suppress excessive repair of the blood vessel walls that were damagedby balloon, so that recurrence of angiostenosis can be prevented.

[0280] In this embodiment, Photofrin® is carried within the drug carrieras a drug for treatment of affected areas, and the ultrasound-sensitivesubstance contained in the drug carriers used to rupture these carriersmay be selected arbitrarily, but Photofrin® may also be used as thisultrasound-sensitive substance.

[0281] Note that examples of drugs for treatment of affected areas thatcan be used in blood vessel therapy include genes, heparin, radioactivesubstances and the like which are administered to the inside wall of theblood vessels.

[0282] Use as a Hemostatic Agent

[0283] Current therapies for liver cancer include injecting ethanol intothe blood vessels that supply nutrition to the cancerous tissue, causingdamage to the inside walls of these blood vessels and thus artificiallycreate thrombi that block these blood vessels, injecting a specific typeof fluid to block the blood vessels and other methods to prevent thepropagation of cancerous cells.

[0284] An alternative method involves preparing Rose Bengal-containingdrug carriers that exhibit action as a blood vessel inside wall damagingagent when activated by ultrasound, and also preparing drug carriersthat contain thrombin which acts as a blood coagulating agent, andinjecting both of these simultaneously into the blood vessel andirradiating the affected area with ultrasound.

[0285] In this embodiment, Rose Bengal is carried within the drugcarriers as the drug for treatment of affected areas. Moreover, theultrasound-sensitive substance contained in the drug carriers used torupture these carriers may be selected arbitrarily, but Rose Bengal mayalso be used.

[0286] In this method, the drug carriers are ruptured by ultrasound torelease the Rose Bengal inside into the affected area, and at this time,the Rose Bengal itself is activated by ultrasound and damages the bloodvessel walls to form thrombi. At the same time the thrombin releasedfrom individual drug carriers causes the blood to coagulate. Therefore,blood flow is halted in the affected area. By combining these two typesof drug carriers in this manner, a synergistic effect as a hemostaticagent is obtained.

[0287] The method described above can be applied not only to thetreatment of liver cancer but also to the stanching of hemorrhage fromorgans due to traffic accidents and the like.

[0288] Transdermal Administration of Medication

[0289] The transdermal administration of medication with the additionaluse of ultrasound is already known. For example, as recited in thespecification of Japanese Patent Application No. 9-166334, a treatmentapparatus consisting of a disc-shaped plate which has a large number offine permeation holes and which contains fluid in the interior wasdeveloped. In this apparatus, the cavitation generation phenomenon canbe used to open fine holes in the surface of skin, so the administrationof medication or collection of bodily fluids can be performedeffectively without any accompanying pain.

[0290] In this embodiment, drug carriers containing anultrasound-sensitive substance are disposed in the permeation holes inthe treatment apparatus described above. Here follows a description inreference to FIG. 29.

[0291] In this preferred embodiment, the transdermal medicationadministration apparatus 140 consists of a film of synthetic resinmaterial or the like which is relatively thin with a thickness in therange of 1 μm-1 cm, where a circular space 142 is formed in theinterior.

[0292] Formed in the bottom-side film 144 of the transdermaladministration apparatus 140 is a plurality of permeation holes 146 thatconnect the circular space 142 to the outside. The diameter of thepermeation holes 144 may be set in the range from 0.1 μm to 3 mm. Notethat in the example shown in FIG. 29, the permeation holes 146 aredistributed uniformly, but they may be provided with nonuniform densityif necessary. In addition, the sectional shape of the permeation holes146 is not limited to circular, as they may also be star-shaped,polygonal-shaped or irregularly shaped. The density of permeation holes146 can be set in the range from 1 to 1 million per square centimeter.

[0293] An ultrasonic oscillator 150 is attached to the top-side film 148of the transdermal administration apparatus 140. This ultrasonicoscillator 150 may be formed as a unit with the transdermaladministration apparatus 140, or a separate ultrasonic oscillator may beprepared independent of the apparatus and pressed against the top-sidefilm 148 of the transdermal administration apparatus 140.

[0294] When the transdermal medication administration apparatus 140 isin use, drug carriers 152 are disposed within the permeation holes 146.The circular space 142 and carrying space 154 in the interior of thedrug carriers 152 are filled with liquid medication 156.

[0295] The bottom-side film 144 of the transdermal administrationapparatus 140 is pressed against the surface of the skin and a drivingsignal is supplied to the ultrasonic oscillator 150 to generateultrasound. Upon doing this, the drug carriers 152 are ruptured and thepermeation holes 146 are opened and at the same time, cavitation occursin the liquid medication 156 within the circular space 142, so a fastflow of fluid that occurs at the time of cavitation collapse passesthrough the permeation holes 146 to reach the skin, forming fine holesin its surface. The liquid medication 156 passes through these holes andis absorbed into the body.

[0296] In this manner, the liquid medicine does not flow out from thepermeation holes 146 while the transdermal administration apparatus 140is in storage, even when the permeation holes 146 are relatively large.On the other hand, the drug carriers 152 are ruptured by ultrasound sothe permeation holes 146 can be opened reliably at the time of use.

[0297] In addition, the ultrasound-sensitive substance inside the drugcarriers 152 lowers the threshold value for the occurrence ofcavitation, so cavitation can be caused to occur in the liquidmedication 156 at a low ultrasonic energy. Thereby the ultrasonic energyabsorbed by the skin can be reduced and the risk of causing deleteriouseffects on the skin is reduced.

[0298] The medications and substances that can be administeredtransdermally using this apparatus include anti-allergic drugs, insulin,various hormones, anti-cancer drugs, anti-inflammatory drugs,anesthetics, anticoagulant factors (heparin, urokinase), antibiotics,various vitamins, steroids, antihypertensive agents, vasopressor drugs,tranquilizers, hair restoratives depilatories and others.

[0299] Treatment of Infectious Disease

[0300] Although the disinfectant effects of UV light are well known, theability of UV light to pass through liquids is extremely poor, so itsintensity attenuates immediately, and thus it is used exclusively forsterilizing the surfaces of medical equipment and the like in air.

[0301] Now the ultrasound-sensitive substance Rose Bengal is known alsoto have the action of reducing the threshold for generation ofcavitation by ultrasound. Taking advantage of this characteristic,ultrasound can be used for the treatment of infectious disease bothinside and outside the body, since ultrasound can be used for thetreatment of infectious disease inside the body.

[0302] To wit, in the treatment of infectious disease inside the body,carriers containing Rose Bengal are infused deeply into the affectedarea by injection or other method, and when the affected area isirradiated with ultrasound in this state, cavitation is generated in thevicinity of these carriers with a relatively low ultrasonic energy, andUV light is generated at the time of its collapse as describedpreviously.

[0303] Therefore, affected areas within the body can be disinfected byirradiating the affected areas with UV light from a very close distance,so this method can be applied to the treatment of infectious disease. Inaddition, this method has an additional advantage in that there is noneed to use various types of antibiotics, so no drug-resistant bacteriaare created.

[0304] Next, in the case of an infectious disease of the skin, drugcarriers that carry a skin absorption accelerant and have their surfacecovered with Rose Bengal are applied to the surface of the skin. RoseBengal permeates the skin relatively easily so the drug carrierspermeate slightly into the surface portion of the skin. If the skin isirradiated with ultrasound in this state, the skin absorption accelerantis released into the skin and the skin's barrier function is lowered orvanishes, so insulin or other drugs that are normally not easilyabsorbed by the skin are absorbed into the skin.

[0305] Note that the above method can be applied not only to thetreatment of infectious diseases of the skin, but alos to the treatmentof athlete's foot, viral blisters, psoriasis scabies, skin cancer,A/DS-related Kaposi's sarcoma and the like.

[0306] Treatment of Diabetes

[0307] The treatment of diabetes can be performed by intravenouslyinjecting drug carriers containing insulin and irradiating the interiorof the body with ultrasound when needed so that these drug carriers areruptured to release the insulin inside into the body. In this case, byadjusting the time and intensity of the irradiation of ultrasound,insulin can be administered regularly with a simple operation.

[0308] In addition, it is possible to use red blood cells in the bloodas the drug carriers described above. For example, red blood cells canbe separated from blood and insulin injected into the individual redblood cells and then the ultrasound-sensitive substance Photofrin® madeto adhere to the surface of the red blood cell membranes.

[0309] By supplying red blood cells treated in this manner into the bodyof the patient through a transfusion or the like, the red blood cellshave a lifetime of approximately 10-100 days, so they will not beruptured during this period unless irradiated with ultrasound, but theinsulin can be released when necessary by irradiation with ultrasoundfrom outside the body. In this case, since the drug carriers consist ofmaterials that are very compatible with the body, namely red bloodcells, rejection from the body can be suppressed.

[0310] As discussed above, the light activated drug can be coupled witha site directing molecule to form a light activated drug conjugate.Suitable site-directing molecules include, but are not limited to:polydeoxyribonucleotides, oligodeoxyribonucleotides, polyribonucleotideanalogs, oligoribonucleotide analogs; polyamides including peptideshaving an affinity for a biological receptor and proteins such asantibodies; steroids and steroid derivatives; hormones such as estradiolor histamine; hormone mimics such as morphine and further macrocyclessuch as sapphyrins and rubyrins. It is understood that the terms“nucleotide”, “polynucleotide”, and “oligonucleotide”, as used hereinand in the appended claims, refer to both naturally occurring andsynthetic nucleotides, poly- and oligonucleotides and to analogs andderivatives thereof such as methylphosphonates, phosphotriesters,phosphorothioates, and phosphoramidates and the like.Deoxyribonucleotides and ribonucleotide analogs are contemplated assite-directing molecules.

[0311] When the site-directing molecule is an oligonucleotide, theoligonucleotide may be derivatized at the bases, the sugars, the end ofthe chains, or at the phosphate groups of the backbone to promote invivo stability. Modifications of the phosphate groups are preferred inone embodiment since phosphate linkages are sensitive to nucleaseactivity. Preferred derivatives are the methylphosphonates,phosphotriesters, phosphorothioates, and phosphoramidates. Additionally,the phosphate linkages may be completely substituted with non-phosphatelinkages such as amide linkages. Appendages to the ends of theoligonucleotide chains also provide exonuclease resistance. Sugarmodifications may include alkyl groups attached to an oxygen of a ribosemoiety in a ribonucleotide. In particular, the alkyl group is preferablya methyl group and the methyl group is attached to the 2′ oxygen of theribose. Other alkyl groups may be ethyl or propyl.

[0312] A linker may be used to couple the light activated drug with thesite directing molecule. Exemplary linkers include, but are not limitedto, amides, amine, thioether, ether, or phosphate covalent bonds asdescribed in the examples for attachment of oligonucleotides. In apreferred embodiment, an oligonucleotide or other site-directingmolecules is covalently bonded to a texaphyrin or other light activateddrugs via a carbon-nitrogen, carbon-sulfur, or a carbon-oxygen bond.

[0313] As described above, the media can be an emulsion which includes alight activated drug. The emulsions described below are suitable fordelivery into a body since they avoid pharmaceutically undesirableorganic solvents, solubilizers, oils or emulsifiers. A wide range oflight activated drug concentrations can be used in the emulsion.Suitable concentrations of light activated drug within the emulsioninclude, but are not limited to, approximately 0.01 to 1 gram/100 ml,preferably about 0.05 to about 0.5 gram/100 ml, and approximately 0.1g/100 ml.

[0314] The emulsion includes a lipoid as a hydrophobic componentdispersed in a hydrophilic phase. The hydrophobic component of theemulsion comprises a pharmaceutically acceptable triglyceride, such asan oil or fat of a vegetable or animal nature, and preferably isselected from the group consisting of soybean oil, safflower oil, marineoil, black current seed oil, borage oil, palm kernel oil, cotton seedoil, corn oil, sunflower seed oil, olive oil or coconut oil. Physicalmixtures of oils and/or interesterfied mixtures can be employed. Thepreferred oils are medium chain length triglycerides having C₈-C₁₀ chainlength and more preferably saturated. The preferred triglyceride is adistillate obtained from coconut oil. The hydrophobic content of theemulsion is preferably approximately 5 to 50 g/100 ml, more preferablyabout 10 to about 30 g/100 ml and approximately 20 g/100 ml of theemulsion.

[0315] The emulsion can also contains a stabilizer such as phosphatides,soybean phospholipids, nonionic block copolymers of polyoxethylene andpolyoxpropylene (e.g., poloxamers), synthetic or semi-syntheticphospholipids, and the like. The preferred stabilizer is purified eggyolk phospholipid. The stabilizer is usually present in the compositionin amounts of about 0.1 to about 10, and preferably about 0.3 to about 3grams/100 ml, a typical example being about 1.5 grams/100 ml.

[0316] The emulsion can also include one or more bile acids salts as acostablizer. The salts are pharmacologically acceptable salts of bileacids selected from the group of cholic acid, deoxycholic acid andgylcocholic acid, and preferably of cholic acid. The salts are typicallyalkaline metal or alkaline earth metal salts and preferably sodium,potassium, calcium or magnesium salts, and most preferably, sodiumsalts. Mixtures of bile acid salts can be employed if desired. Theamount of bile acid salt employed is usually about 0.01 to about 1.0 andpreferably about 0.05 to about 0.4 grams/100 ml, a typical example beingabout 0.2 grams/100 ml.

[0317] Suitable pH for the emulsion includes, but is not limited toapproximately 7.5 to 9.5, and preferably approximately 8.5. The pH canbe adjusted to the desired value, if necessary, by adding apharmaceutically acceptable base, such as sodium hydroxide, potassiumhydroxide, calcium hydroxide, magnesium hydroxide and ammoniumhydroxide.

[0318] Water can be added to the emulsion to achieve the desiredconcentration of various components within the emulsion. Further, theemulsion can include auxiliary ingredients for regulating the osmoticpressure to make the emulsion isotonic with the blood. Suitableauxiliary ingredients include, but are not limited to, auxiliarysurfactants, isotonic agents, antioxidants, nutritive agents, traceelements and vitamins. Suitable isotonic agents include, but are notlimited to, glycerin, amino acids, such as alanine, histidine, glycine,and/or sugar alcohols, such as xylitol, sorbitol and/or mannitol.Suitable concentrations for isotonic agents within the emulsion include,but are not limited to, approximately 0.2 to about 8.0 grams/100 ml andpreferably about 0.4 to about 4 grams/100 ml and most preferably 1.5 to2.5 gram/100 ml.

[0319] Antioxidants can be used to enhance the stability of theemulsion, a typical example being α-tocopherol. Suitable concentrationsfor the antioxidants include, but are not limited to approximately 0.005to 0.5 grams/100 ml, approximately 0.02 to about 0.2 grams/100 ml andmost preferably approximately 0.05 to 0.15 grams/100 ml.

[0320] The emulsions can also contain auxiliary solvents, such as analcohol, such as ethyl alcohol or benzyl alcohol, with ethyl alcoholbeing preferred. When employed, such is typically present in amounts ofabout 0.1 to about 4.0, and preferably about 0.2 to about 2 grams/100ml, a typical example being about 1 gram/100 ml. The ethanol isadvantageous since it facilitates dissolution of poorly water-solublelight activated drugs and especially those that form crystals which maybe very difficult to dissolve in the hydrophobic phase. Accordingly, theethanol must be added directly to the hydrophobic phase duringpreparation to be effective. For maximum effectiveness, the ethanolshould constitute about 5% to 15% by weight of the hydrophobic phase. Inparticular, if ethanol constitutes less than 5% by weight of thehydrophobic phase, dissolution of the light activated drug can becomeunacceptably slow. When the ethanol concentration exceeds 15%, large (>5μm diameter) and poorly emulsified oil droplets can form in theemulsion. The particles in the emulsion are preferably less than about5.0 μm in diameter, more preferably less than 2.0 μm in diameter andmost preferably less than 0.5 μm or below.

[0321] A typical emulsion is prepared using the following technique. Thetriglyceride oil is heated to 50°-70° C. while sparging with nitrogengas. The required amounts of stabilizer (e.g., egg yolk phospholipids),bile acid salt, alcohol (e.g., ethanol), antioxidant (e.g.,α-to-copherol) and light activated drug are added to the triglyceridewhile processing for about 5 to about 20 minutes with a high speedblender or overhead mixer to ensure complete dissolution or uniformsuspension.

[0322] In a separate vessel, the required amounts of water and isotonicagent (e.g., -glycerin) are heated to the above temperature (e.g.,50°-70°) while sparging with nitrogen gas. Next, the aqueous phase istransferred into the prepared hydrophobic phase and high speed blendingis continued for another 5 to 10 minutes to produce a uniform but coarsepreemulsion (or premix). This premix is then transferred to aconventional high pressure homogenizer (APV Gaulin) for emulsificationat about 8,000-10,000 psi. The diameter of the dispersed oil droplets inthe finished emulsion will be less than 5 μm, with a large proportionless than 1 μm. The mean diameter of these oil droplets will be lessthan 1 μm, preferably from 0.2 to 0.5 μm. The emulsion product is thenfilled into borosilicate (Type 1) glass vials which are stoppered,capped and terminally heat sterilized in a rotating steam autoclave atabout 121° C.

[0323] These emulsions can withstanding autoclaving as well as freezingat about 0° to −20° C. Such can be stored for a relatively long timewith minimal physical and chemical breakdown, i.e. at least 12-18 monthsat 4°-8° C. The vehicle composition employed is chemically inert withrespect to the incorporated pharmacologically active light activateddrug.

[0324] The emulsions can exhibit very low toxicity following intravenousadministration and exhibit no venous irritation and no pain oninjection. The emulsions exhibit minimal physical and chemical changes(e.g., formation of non-emulsified surface oil) during controlledshake-testing on a horizontal platform. Moreover, the oil-in-wateremulsions promote desirable pharmacoldnetics and tissue distribution ofthe light activated drug in vivo.

[0325] As discussed above, the light activated drug can also bedelivered to the body in a media which includes microbubbles. Suitablesubstrates for the microbubble include, but are not limited to,biocornipatible polymers, albumins, lipids, sugars or other substances.U.S. Pat. Nos. 5,701,899 and 5,578,291 teach a method for synthesizingmicrobubbles with a sugar and protein substrate and is incorporatedherein by reference. U.S. Pat. Nos. 5,665,383 and 5,665, 382 teaches amethod for synthesizing microbubbles with a polymeric substrate and isincorporated herein by reference. U.S. Pat. Nos. 5,626,833 and 5,798,091teach methods for synthesizing microbubbles with a surfactant substrateand are incorporated herein by reference. A preferred microbubble has alipid substrate. U.S. Pat. Nos. 5,772,929 teaches methods forsynthesizing microbubbles with a lipid substrate. U.S. Pat. Nos.5,776,429, 5,715,824 and 5,770,222 teach preferred methods forsynthesizing microbubbles with a lipid substrate and a gas interior andare incorporated herein by reference.

[0326] Suitable microbubbles with a lipid substrate can be liposomes.The liposomes can be unilamellar vesicles having a single membranebilayer or multilamellar vesicles having multiple membrane bilayers,each bilayer being separated from the next by an aqueous layer. Aliposome bilayer is composed of two lipid monolayers having ahydrophobic “tail” region and a hydrophilic “head” region. The formulaof the membrane bilayer is such that the hydrophobic (nonpolar) “tails”of the lipid monolayers orient themselves towards the center of thebilayer, while the hydrophilic “heads” orient themselves toward theaqueous phase. Either unilamellar or multilamellar or other types ofliposomes may be used.

[0327] A hydrophilic light activated drug can be entrapped in theaqueous phase of the liposome before the drug is delivered into thepatient. Alternatively, if the light activated drug is lipophilic, itmay associate with the lipid bilayer. Liposomes may be used to help“target” the light activated drug to an active site or to solubilizehydrophobic light activated drugs. Light activated drugs are typicallyhydrophobic and form stable drug-lipid complexes.

[0328] As discussed above, many light activated drugs have lowsolubility in water at physiological pH's, but are also insoluble in (1)pharmaceutically acceptable aqueous-organic co-solvents, (2) aqueouspolymeric solutions and (3) surfactant/micellar solutions. However, suchlight activated drugs can still be “solubilized” in a form suitable fordelivery into a body by using a liposome composition. For example, oneexample of a light activated drug BPD-MA (See Formula A of FIG. 20) canbe “solubilized” at a concentration of about 2.0 mg/ml in aqueoussolution using an appropriate mixture of phospholipids to formencapsulating liposomes.

[0329] Although the light activated drug can be included in manydifferent types of liposomes, the following description disclosesparticular liposome compositions and methods for making the liposomeswhich are known to be “fast breaking”. In fast breaking liposomes, thelight activated drug-liposome combination is stable in vitro but, whenadministered in vivo, the light activated drug is rapidly released intothe bloodstream where it can associate with serum lipoproteins. As aresult, the localized delivery of liposomes combined with the fastbreaking nature of the liposomes can result in localization of the lightactivated drug in the tissues near the catheter. Further, the fastbreaking liposomes can prevent the liposomes from leaving the vicinityof the catheter intact and then concentrating in non-targeted tissuessuch as the liver. Delivery of ultrasound energy from the catheter canalso serve to break apart the liposomes after they have been deliveredfrom the catheter.

[0330] Liposomes are typically formed spontaneously by adding water to adry lipid film. Liposomes which include light activated drugs caninclude a mixture of the commonly encountered lipids dimyristoylphosphatidyl choline (“DMPC”) and egg phosphatidyl glycerol (“EPG”). Thepresence of DMPC is important because DMPC is the major component in thecomposition to form liposomes which can solubilize and encapsulateinsoluble light activated drugs into a lipid bilayer. The presence ofEPG is important because the negatively charged, polar head group ofthis lipid can prevent aggregation of the liposomes.

[0331] Other phospholipids, in addition to DMPC and EPG, may also bepresent. Examples of suitable additional phospholipids that may also beincorporated into the liposomes include phosphatidyl cholines (PCS),including mixtures of dipalmitoyl phosphatidyl choline (DPPC) anddistearoyl phosphatidyl choline (DSPC). Examples of suitablephosphatidyl glycerols (PGs) include dimyristoyl phosphatidyl glycerol(DMPG), DLPG and the like.

[0332] Other types of suitable lipids that may be included arephosphatidyl ethanolamines (PEs), phosphatidic acids (PAs), phosphatidylserines, and phosphatidyl inositols.

[0333] The molar ratio of the light activated drug to the DMPC/EPGphospholipid mixture can be as low as 1:7.0 or may contain a higherproportion of phospholipid, such as 1:7.5. Preferably, this molar ratiois 1:8 or more phospholipid, such as 1:10, 1:15, or 1:20. This molarratio depends upon the exact light activated drug being used, but willassure the presence of a sufficient number of DMPC and EPG lipidmolecules to form a stable complex with many light activated drugs. Whenthe number of lipid molecules is not sufficient to form a stablecomplex, the lipophilic phase of the lipid bilayer becomes saturatedwith light activated drug molecules. Then, any slight change in theprocess conditions can force some of the previously encapsulated lightactivated drug to leak out of the vesicle, onto the surface of the lipidbilayer, or even out into the aqueous phase.

[0334] If the concentration of light activated drug is high enough, itcan actually precipitate out from the aqueous layer and promoteaggregation of the liposomes. The more unencapsulated light activateddrug that is present, the higher the degree of aggregation. The moreaggregation, the larger the mean particle size will be, and the moredifficult aseptic or sterile filtration will be. As a result, smallchanges in the molar ratio can be important in achieving properfilterability of the liposome composition.

[0335] Accordingly, slight increases in the lipid content can increasesignificantly the filterability of the liposome composition byincreasing the ability to form and maintain small particles. This isparticularly advantageous when working with significant volumes of 500ml, a liter, five liters, 40 liters, or more, as opposed to smallerbatches of about 100-500 ml or less. This volume effect is thought tooccur because larger homogenizing devices tend to provide less efficientagitation than can be accomplished easily on a small scale. For example,a large size Microfluidizer™ has a less efficient interaction chamberthan that one of a smaller size.

[0336] A molar ratio of 1.05:3:5 BPD-MA:EPG:DMPC (i.e., slightly lessphospholipid than 1:8.0 light activated drug:phospholipid) may providemarginally acceptable filterability in small batches of up to 500 ml.However, when larger volumes of the composition are being made, a highermolar ratio of phospholipid provides more assurance of reliable asepticfilterability. Moreover, the substantial potency losses that are commonin scale-up batches, due at least in part to filterability problems, canthus be avoided.

[0337] Any cryoprotective agent known to be useful in the art ofpreparing freeze-dried formulations, such as di- or polysaccharides orother bulking agents such as lysine, may be used. Further, isotonicagents typically added to maintain isomolarity with body fluids may beused. In a preferred embodiment, a di-saccharide or polysaccharide isused and functions both as a cryoprotective agent and as an isotonicagent.

[0338] In a particular embodiment, the particular combination of thephospholipids, DMPC and EPG, and a disaccharide or polysaccharide form aliposomal composition having liposomes of a particularly narrow particlesize distribution. When the process of hydrating a lipid film isprolonged, larger liposomes tend to be formed, or the light activateddrug can even begin to precipitate. The addition of a disaccharide orpolysaccharide provides instantaneous hydration and the large surfacearea for depositing a thin film of the drug-phospholipid complex. Thisthin film provides for faster hydration so that, when the liposome isinitially formed by adding the aqueous phase, the liposomes formed areof a smaller and more uniform particle size. This provides significantadvantages in terms of manufacturing ease.

[0339] However, it is also possible that, when a saccharide is presentin the composition, it is added after dry lipid film formation, as apart of the aqueous solution used in hydration. In a particularlypreferred embodiment, a saccharide is added to the dry lipid film duringhydration.

[0340] Disaccharides or polysaccharides are preferred to monosaccharidesfor this purpose. To keep the osmotic pressure of the liposomecomposition similar to that of blood, no more than 4-5% monosaccharidescould be added. In contrast, about 9-10% of a disaccharide can be usedwithout generating an unacceptable osmotic pressure. The higher amountof disaccharide provides for a larger surface area, which results insmaller particle sizes being formed during hydration of the lipid film.

[0341] Accordingly, the preferred liposomal composition comprises adisaccharide or polysaccharide, in addition to the light activated drugand the mixture of DMPC and EPG phospholipids. When present, thedisaccharide or polysaccharide is preferably chosen from among the groupconsisting of lactose, trehalose, maltose, maltotriose, palatinose,lactulose or sucrose, with lactose or trehalose being preferred. Evenmore preferably, the liposomes comprise lactose or trehalose.

[0342] Also, when present, the disaccharide or polysaccharide isformulated in a preferred ratio of about 10-20 saccharide to 0.5-6.0DMPC/EPG phospholipid mixture, respectively, even more preferably at aratio from about 10 to 1.5-4.0. In one embodiment, a preferred but notlimiting formulation is lactose or trehalose and a mixture of DMPC andEPG in a concentration ratio of about 10 to 0.94-1.88 to about0.65-1.30, respectively.

[0343] The presence of the disaccharide or polysaccharide in thecomposition not only tends to yield liposomes having extremely small andnarrow particle size ranges, but also provides a liposome composition inwhich light activated drugs, in a particular, may be stably incorporatedin an efficient manner, i.e., with an encapsulation efficiencyapproaching 80-100%. Moreover, liposomes made with a saccharidetypically exhibit improved physical and chemical stability, such thatthey can retain an incorporated light activated drug without leakageupon prolonged storage, either as a reconstituted liposomal or as acryodesiccated powder.

[0344] Other optional ingredients include minor amounts of nontoxic,auxiliary substances in the liposomal composition, such as antioxidants,e.g., butylated hydroxytoluene, alphatocopherol and ascorbyl palmitate;pH buggering agents, e.g., phosphates, glycine, and the like.

[0345] Liposomes containing a light activated drug may be prepared bycombining the light activated drug and the DMPC and EPG phospholipids(and any other optional phospholipids or excipients, such asantioxidants) in the presence of an organic solvent. Suitable organicsolvents include any volatile organic solvent, such as diethyl ether,acetone, methylene chloride, chloroform, piperidine, piperidine-watermixtures, methanol, tert-butanol, dimethyl sulfoxide,N-methyl-2-pyrrolidone, and mixtures thereof. Preferably, the organicsolvent is water-immiscible, such as methylene chloride, but waterimmiscibility is not required. In any event, the solvent chosen shouldnot only be able to dissolve all of the components of the lipid film,but should also not react with, or otherwise deleteriously affect, thesecomponents to any significant degree.

[0346] The organic solvent is then removed from the resulting solutionto form a dry lipid film by any known laboratory technique that is notdeleterious to the dry lipid film and the light activated drug.Preferably, the solvent is removed by placing the solution under avacuum until the organic solvent is evaporated. The solid residue is thedry lipid film. The thickness of the lipid film is not critical, butusually varies from about 30 to about 45 mg/cm² depending upon theamount of solid residual and the total area of the glass wall of theflask. Once formed, the film may be stored for an extended period oftime, preferably not more than 4 to 21 days, prior to hydration. Whilethe temperature during a lipid film storage period is also not animportant factor, it is preferably below room temperature, mostpreferably in the range from about −20 to about 4° C.

[0347] The dry lipid film is then dispersed in an aqueous solution,preferably containing a disaccharide or polysaccharide, and homogenizedto form the desired particle size. Examples of useful aqueous solutionsused during the hydration step include sterile water; a calcium- andmagnesium-free, phosphate-buffered (pH 7.2-7.4) sodium chloridesolution; a 9.75% w/v lactose solution; a lactose-saline solution; 5%dextrose solution; or any other physiologically acceptable aqueoussolution of one or more electrolytes. Preferably, however, the aqueoussolution is sterile. The volume of aqueous solution used duringhydration can vary greatly, but should not be so great as about 98% norso small as about 30-40%. A typical range of useful volumes would befrom about 75% to about 95%, preferably about 85% to about 90%.

[0348] Upon hydration, coarse liposomes are formed that incorporate atherapeutically effective amount of the light activateddrugs-phospholipid complex. The “therapeutically effective amount” canvary widely, depending on the tissue to be treated and whether it iscoupled to a target-specific ligand, such as an antibody or animmunologically active fragment. It should be noted that the variousparameters used for selective photodynamic therapy are interrelated.Therefore, the therapeutically effective amount should also be adjustedwith respect to other parameters, for example, fluence, irradiance,duration of the light used in photodynamic therapy, and the timeinterval between administration of the light activated drug and thetherapeutic irradiation. Generally, all of these parameters are adjustedto produce significant damage to tissue deemed undesirable, such asneovascular or tumor tissue, without significant damage to thesurrounding tissue, or to enable the observation of such undesirabletissue without significant damage to the surrounding tissue.

[0349] Typically, the therapeutically effective amount is such toproduce a dose of light activated drug within a range of from about 0.1to about 20 mg/kg, preferably from about 0.15-2.0 mg/kg and, even morepreferably, from about 0.25 to about 0.75 mg/kg. Preferably, the w/vconcentration of the light activated drug in the composition ranges fromabout 0.1 to about 8.0-10.0 g/L. Most preferably, the concentration isabout 2.0 to 2.5 g/L.

[0350] The hydration step should take place at a temperature that doesnot exceed about 30° C., preferably below the glass transitiontemperature of the light activated drug-phospholipid complex formed,even more preferably at room temperature or lower, e.g., 15°-20° C. Theglass transition temperature of the light activated drug-lipid complexcan be measured by using a differential scanning microcalorimeter.

[0351] In accordance with the usual expectation that the aqueoussolubility of a substance should increase as higher temperatures areused, at a temperature around the transition temperature of the complex,the lipid membrane tends to undergo phase transition from a “solid” gelstate to a pre-transition state and, finally, to a more “fluid” liquidcrystal state. At these higher temperatures, however, not only doesfluidity increase, but the degree of phase separation and the proportionof membrane defects also increases. This results in an increasing degreeof leakage of the light activated drug from inside the membrane to theinterface and even out into the aqueous phase. Once a significant amountof liposome leakage has occurred, even slight changes in the conditionssuch as a small drop in temperature, can shift the equilibrium away fromaqueous “solubility” in favor of precipitation of the light activateddrug. Moreover, once the typically water-insoluble light activated drugbegins to precipitate, it is not possible to re-encapsulate it when thelipid bilayer. The precipitate is thought to contribute significantly tofilterability problems.

[0352] In addition, the usual thickness of a lipid bilayer in the“solid” gel state (about 47 A) decreases in the transition to the“liquid” crystalline state to about 37 A, thus shrinking the entrappedvolume available for the light activated drugs to occupy. The smaller“room” is not capable of containing as great a volume of light activateddrug, which can then be squeezed out of the saturated lipid bilayerinterstices. Any two or more liposomes exuding light activated drug mayaggregate together, introducing further difficulties with respect toparticle size reduction and ease of sterile filtration. Moreover, theuse of higher hydration temperatures, such as, for example, about 35° to45° C., can also result in losses of light activated drug potency as thelight activated drug either precipitates or aggregates during asepticfiltration.

[0353] The particle sizes of the coarse liposomes first formed inhydration are then homogenized to a more uniform size, reduced to asmaller size range, or both, to about 150 to 300 nm, preferably also ata temperature that does not exceed about 30° C., preferably below theglass transition temperature of the light activated drug-phospholipidcomplex formed in the hydration step, and even more preferably belowroom temperature of about 25° C. Various high-speed agitation devicesmay be used during the homogenization step, such as a Microfluidizer™model 110F; a sonicator; a high-shear mixer; a homogenizer; or astandard laboratory shaker

[0354] It has been found that the homogenization temperature should beat room temperature or lower, e.g., 15°-20° C. At higher homogenizationtemperatures, such as about 32°-42° C., the relative filterability ofthe liposome composition may improve initially due to increased fluidityas expected, but then, unexpectedly, tends to decrease with continuingagitation due to increasing particle size.

[0355] Preferably, a high pressure device such a Microfluidizer™ is usedfor agitation. In microfluidization, a great amount of heat is generatedduring the short-period of time during which the fluid passes through ahigh pressure interaction chamber. In the interaction chamber, twostreams of fluid at a high speed collide with each other at a 90° angle.As the microfluidization temperature increases, the fluidity of themembrane also increases, which initially makes particle size reductioneasier, as expected. For example, filterability can increase by as muchas four times with the initial few passes through a Microfluidizer™device. The increase in the fluidity of the bilayer membrane promotesparticle size reduction, which makes filtration of the final compositioneasier. In the initial several passes, this increased fluidity mechanismadvantageously dominates the process.

[0356] However, as the number of passes and the temperature bothincrease, more of the hydrophobic light activated drug molecules aresqueezed out of the liposomes, increasing the tendency of the liposomesto aggregate into larger particles. At the point at which theaggregation of vesicles begins to dominate the process, the sizes cannotbe reduced any further. Surprisingly, particle sizes actually then tendto grow through aggregation.

[0357] For this reason, the homogenization temperature is cooled down toand maintained at a temperature no greater than room temperature afterthe composition passes through the zone of maximum agitation, e.g., theinteraction chamber of a Microfluidizer™ device. An appropriate coolingsystem can easily be provided for any standard agitation device in whichhomogenization is to take place, e.g., a Microfluidizer™, such as bycirculating cold water into an appropriate cooling jacket around themixing chamber or other zone of maximum turbulence. While the pressureused in such high pressure devices is not critical, pressures from about10,000 to about 16,000 psi are not uncommon.

[0358] As a last step, the compositions are preferably asepticallyfiltered through a filter having an extremely small pore size, i.e.,0.22 μm. Filter pressures used during sterile filtration can varywidely, depending on the volume of the composition, the density, thetemperature, the type of filter, the filter pore size, and the particlesize of the liposomes. However, as a guide, a typical set of filtrationconditions would be as follows: filtration pressure of 15-25 psi;filtration load of 0.8 to 1.5 ml/cm²; and filtration temperature ofabout 25° c.

[0359] A typical general procedure is described below with additionalexemplary detail:

[0360] (1) Sterile filtration of organic solvent through a hydrophobic,0.22 μm filter.

[0361] (2) Addition of EPG, DMPC, light activated drug, and excipientsto the filtered organic solvent, dissolving both the excipients and thelight activated drug.

[0362] (3) Filtration of the resulting solution through a 0.22 μmhydrophobic filter.

[0363] (4) Transfer of the filtrate to a rotary evaporator apparatus,such as that commercially available under the name Rotoevaporator™.

[0364] (5) Removal of the organic solvent to form a dry lipid film.

[0365] (6) Analysis of the lipid film to determine the level of organicsolvent concentration.

[0366] (7) Preparation of a 10% lactose solution.

[0367] (8) Filtration of the lactose solution through a 0.22 μmhydrophilic filter.

[0368] (9) Hydration of the lipid film with a 10% lactose solution toform coarse liposomes.

[0369] (10) Reduction of the particle sizes of the coarse liposomes bypassing them through a Microfluidizer™ three times.

[0370] (11) Determination of the reduced particle size distribution ofliposomes.

[0371] (12) Aseptic filtration of the liposome composition through a0.22 μm hydrophilic filter. (Optionally, the solution may first bepre-filtered with a 5.0 μm prefilter.)

[0372] (13) Analysis of light activated drug potency.

[0373] (14) Filling of vials with the liposome composition.

[0374] (15) Freeze-drying.

[0375] Once formulated, the liposome composition may be freeze-dried forlong-term storage if desired. For example, BPD-MA, a preferred lightactivated drug, has maintained its potency in a cryodesiccated liposomecomposition for a period of at lest nine months at room temperature, anda shelf life of at least two years has been projected. If thecomposition is freeze-dried, it may be packed in vials for subsequentreconstitution with a suitable aqueous solution, such as sterile wateror sterile water containing a saccharide and/or other suitableexcipients, prior to administration, for example, by injection.

[0376] Preferably, liposomes that are to be freeze-dried are formed uponthe addition of an aqueous vehicle contain a disaccharide orpolysaccharide during hydration. The composition is then collected,placed into vials, freeze-dried, and stored, ideally underrefrigeration. The freeze-dried composition can then be reconstituted bysimply adding water for injection just prior to administration.

[0377] The liposomal composition provides liposomes of a sufficientlysmall and narrow particle size that the aseptic filtration of thecomposition through a 0.22 μm hydrophilic filter can be accomplishedefficiently and with large volumes of 500 ml to a liter or more withoutsignificant clogging of the filter. A particularly preferred particlesize range is below about 300 nm, more preferably below from about 250nm. Most preferably, the particle size is below about 220 nm.

[0378] Generally speaking, the concentration of the light activateddrugs in the liposome depends upon the nature of the light activateddrug used. When BPD-MA is used for example, the light activated drug isgenerally incorporated in the liposomes at a concentration of about0.10% up to 0.5% w/v. If freeze-dried and reconstituted, this wouldtypically yield a reconstituted solution of up to about 5.0 mg/ml lightactivated drug.

[0379] For diagnosis, the light activated drugs incorporated intoliposomes may be used along with, or may be labeled with, a radioisotopeor other detecting means. If this is the case, the detection meansdepends on the nature of the label. Scintigraphic labels such astechnetium or indium can be detected using ex vivo scanners. Specificfluorescent labels can also be used but, like detection based onfluorescence of the light activated drugs themselves, these labels canrequire prior irradiation.

[0380] The methods of preparing various light activated drugs, lightactivated drug conjugates, emulsions and microbubbles are described ingreater detail in the examples below. These examples are readily adaptedto preparing analogous light activated drugs, light activated drugconjugates, emulsions and microbubbles by substitutions of appropriatelight activated drugs, site directing molecule, phospholipids, and otheranalogous components. The following examples are being presented todescribe the preferred components, embodiments, utilities and attributesof the media. For example, although BPD-MA is used as the lightactivated drugs in the microbubble (liposome) examples, the invention isnot intended to be limited to this particular light activated drug.

[0381] Example 1 describes the synthesis of a preferred texaphyrinderivative. Examples 2-4 describe different light activated drugsconjugated with oligonucleotides as site directing molecules. Examples 5and 6 describes a synthesis of an emulsion including a light activateddrug. Example 7 describes preparation of microbubbles which include alight activated drug.

EXAMPLE 1 Synthesis of Texaphyrin T2BET Metal Complexes

[0382] The synthesis of texaphyrins is provided in U.S. Pat. Nos.4,935,498, 5,162,509 and 5,252,720, all incorporated by referenceherein. The present example provides the synthesis of a preferredtexaphyrin, named T2BET, having substituents containing ethoxy groups.

[0383] Lutetium(III) acetate hydrate can be purchased from StremChemicals, Inc. (Newburyport, Mass.), gadolinium(III) acetatetetrahydrate can be purchased from Aesar/Johnson Matthey (Ward Hill,Mass.) and LZY-54 zeolite can be purchased from UOP (Des Plaines, Ill.).Acetone, glacial acetic acid, methanol, ethanol, isopropyl alcohol, andn-heptanes can be purchased from J. T. Baker (Phillipsburg, N.J.).Triethylamine and Amberlite 904 anion exchange resin can be purchasedfrom Aldrich (Milwaukee, Wisc.). All chemicals should be ACS grade andused without further purification.

[0384] FIGS. 30A-I illustrate the synthesis of the gadolinium (III)complex of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy) ethoxy]ethoxy]-pentaazapentacyclo[20.2.1.1^(3,6).I^(8,11).0^(14,19)]heptacosa-1,3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridecaene which is illustrated as Formula I ofFIG. 30. The critical intermediate1,2-bis[2-[2-(2-methoxyethoxy)ethoxy)ethoxy]-4,5-dinitrobenzene (FormulaE) can be prepared according to a three-step synthetic process outlinedin FIGS. 30A-I. (Note: References to “Formula A,” “Formula B,” etc.relate to FIGS. 30A, 30B, etc.).

[0385] Synthesis of triethylene glycol monomethyl ether monotosylate,Formula B: In an oven dried 12 L three-necked roundbottom flask,equipped with a magnetic stir bar and a 1000 mL pressure-equalizingdropping funnel, a solution of NaOH (440.0 g, 11.0 mol) is added to 1800mL water and the mixture is cooled to approximately 0° C. A solution oftriethylene glycol monomethyl ether, Formula A, (656.84 g, 4.0 mol) inTHF (1000 mL) is added. The clear solution is stirred vigorously at 0°C. for 15 min and a solution of tosyl chloride (915.12, 4.8 mol) in THF(2.0 L) added dropwise over a 1 h period. The reaction mixture isstirred for an additional 1 h at 0° C., and 10% HCl (5.0 L) is added toquench the reaction (to pH 5-7). The two-phase mixture is transferred toa 4 L separatory funnel, the organic layer removed, and the aqueouslayer extracted with t-butylmethyl ether (3×250 mL). The combinedorganic extracts are washed with brine (2×350 μL), dried (MgSO₄), andevaporated under reduced pressure to afford Formula B, 1217.6 g (95%) asa light colored oil. This material is taken to the next step withoutfurther purification.

[0386] Synthesis of 1,2-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]benzene,Formula D: In a dry 5 L round-bottom flask equipped with an overheadstirrer, reflux condenser, and a gas line, K₂CO₃ (439.47 g, 3.18 mol)and MeOH (1800 mL) are combined under an argon atmosphere. To thiswell-stirred suspension, catechol, Formula C, (140.24 g, 1.21 mol) isadded, and the mixture heated to reflux. Formula B (1012.68 g, 3.18 mol)is then added in one portion. The suspension is stirred at reflux for 24h, cooled to room temperature, and filtered through Celite. The pad isrinsed with 500 mL of methanol and the combined filtrates are evaporatedunder reduced pressure. The resulting brown residue is taken up in 10%NaOH (800 mL), and methylene chloride (800 mL) added with stirring. Themixture is transferred to a 2 L separatory funnel, the organic layerremoved and the aqueous layer extracted with methylene chloride (3×350mL). The organic extracts are combined, washed with brine (350 mL),dried (MgSO₄), evaporated under reduced pressure, and the residue driedin vacuo for several hours to yield 485.6 (95%) of1,2-bis[2-[2-(2-methoxyethoxy)ethoxy)ethoxy]benzene (Formula D). ForFormula D: bp. 165°-220° C., (0.2-0.5 mm Hg); FAB MS, M⁺: m/e 402; HRMS,M⁺: 402.2258 (calcd. for C₂₀H₃₄O₈, 402.2253).

[0387] Synthesis of 1,2-bis[2-[2-(2-methoxyetboxy)ethoxy]ethoxy]-4,5-dinitrobenzene, Formula E: In an oven dried 1 L roundbottom flaskFormula D (104 g, 0.26 mol) and glacial acetic acid (120 mL) arecombined and cooled to 5° C. To this well stirred solution, concentratednitric acid (80 mL) is added dropwise over 15-20 min. The temperature ofthe mixture is held below 40° C. by cooling and proper regulation of therate of addition of the acid. After addition, the reaction is allowed tostir for an additional 10-15 min and is then cooled to 0° C. Fumingnitric acid (260 mL) is added dropwise over 30 min while the temperatureof the solution is held below 30° C. After the addition is complete, thered colored solution is allowed to stir at room temperature until thereaction is complete (ca. 5 h, TLC: 95/5; CH₂Cl₂/MeOH) and then pouredinto well stirred ice water (1500 mL). Methylene chloride (400 mL) isadded, the two-phase mixture transferred to a 2 L separatory funnel andthe organic layer removed. The aqueous layer is extracted with CH₂Cl₂(2×150 mL) and the combined organic extracts washed with 10% NaOH (2×250mL) and brine (250 mL), dried (MgSO₄), and concentrated under reducedpressure. The resulting orange oil is dissolved in acetone (100 mL), andthe solution layered with n-hexanes (500 mL), and stored in the freezer.The resulting precipitate is collected by filtration yield 101.69 g(80%) of Formula E as a yellow solid. For Formula E: mp 43°-45° C.; FABMS, (M+H)⁺: m/e 493; HRMS, (M+H)⁺; 493; HRMS, (M+H)⁺: 493.2030 (calcd.for C₂₀H₃₃N₂O₁₂, 493.2033).

[0388] Synthesis of1,2-diamino-4,5-bis[2-[2-(2-methoxy-ethoxy)ethoxy]ethoxy], Formula F: Inan oven dried 500 mL round bottom flask, equipped with a Claisenadapter, pressure equalizing dropping funnel, and reflux condenser,1,2-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-4,5-dinitrobenzene (FormulaE) (20 g, 0.04 mol) is dissolved in absolute ethanol (200 mL). To thisclear solution, 10% palladium on carbon (4 g) is added and the darkblack suspension is heated to reflux under an argon atmosphere.Hydrazine hydrate (20 mL) in EtOH (20 mL) is added dropwise over 10 minto avoid bumping. The resulting brown suspension is heated at reflux for1.5 h at which time the reaction mixture is colorless and TLC analysis(95/5; CH₂Cl₂/MeOH) displays a low R_UV active spot corresponding to thediamine. Therefore, the mixture is hot filtered through Celite and thepad rinsed with absolute ethanol (50 mL). The solvent is removed underreduced pressure and the resulting light brown oil is dried in vacuo (inthe dark) for 24 h to yield 15.55 g (89%) of1,2-diamino-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]benzene (Formula F).For Formula F: FAB MS,M⁺: m/e 432; HRMS, M⁺: 432.2471 (calcd. forC₂₀H₃₆N₂O₈, 432.2482). This material is taken to the next step withoutfurther purification.

[0389] Synthesis of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo.

[0390] [20.2.1.1^(3,6). 18,11.0^(14,19)]heptacosa-3,5,8,10,12,14,16,18,20,22,24-undecaene, FormulaH. In an oven dried 1 L round-bottom flask,2,5-bis[(5-formyl-3-(3-hydroxypropyl)-4-methyl-pyrrol-2yl)methyl]-3,4-diethylpyrrole(Formula G) (The synthesis of Formula G is provided in U.S. Pat. No.5,252,720, incorporated by reference herein.) (30.94) g, 0.0644 mol) and4,5-diamino-bis[2[2-(2-methoxyetboxy)ethoxy)ethoxy]benzene (Formula F)(28.79 g, 0.0644 mol) are combined in absolute methanol (600 mL) underan argon atmosphere. To this well stirred suspension, a mixture ofconcentrated hydrochloric acid (6.7 m:) in absolute methanol 200 mL isadded in one portion. The mixture is gradually heated to 50° C., atwhich time the reaction goes from a cloudy suspension of startingmaterials to a dark red homogeneous solution as the reaction proceeded.After 3 h the reaction is judged complete by TLC analysis and UV/visiblespectroscopy (λ_(max) 369 nm). The reaction mixture is cooled to roomtemperature, 60 g of activated carbon (DARCO™) is added, and theresulting suspension is stirred for 20 min. The dark suspension isfiltered through Celite to remove the carbon, the solvent evaporated todryness, and the crude Formula H dried in vacuo overnight. Formula H isrecrystallized from isopropyl alcohol/n-heptane to afford 50 g (85%) ofa scarlet red solid. For Formula H: ¹H NMR (CD₃OD): ∂ 1.11 (t, 6H,CH₂CH₃), 1.76 (p, 4H, pyrr-CH₂CH₂CH₂OH), 2.36 (s, 6H, pyrr-CH₃), 2.46(q, 4H, CH₂CH₃), 2.64 (t, 4H, pyrr-CH₂CH₂CH₂OH), 3.29 [s, 6H,(CH₂CH₂O)₃CH₃], 3.31 (t, 4H, pyrr-CH₂CH₂CH₂OH), 3.43-3.85 (m, 20H,CH₂CH₂OCH₂CH₂OCH₂CH₂O), 4.0 (s, 4H, (pyrr)₂—CH₂), 4.22 (t, 4H,PhOCH₂CH₂O), 7.45 (s, 2H, PhH), 8.36 (s, 2H, HC═N); UV/vis:[(MeOH)λ_(max)]: 369; FAB MS, [M+H]⁺: m/e 878.5; HRMS, [M+H]⁺: m/e878.5274 (calcd. for [C₄₈H₇₂N₅O₁₀]⁺ 878.5279).

[0391] Synthesis of the gadolinium (III) complex of4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]pentaazapentacyclo[20.2.1.1^(3,6),1^(8,11),0^(14,19)]heptacosa-1,3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridecaene,Formula I. Formula I is prepared according to the process outlined inFIG. 30. In a dry 2 L three-necked round-bottom flask, Formula H (33.0g, 0.036 mol) and gadolinium(II) acetate tetrahydrate (15.4 g, 0.038mol) are combined in methanol (825 mL). To this well stirred redsolution, gadolinium(III) acetate tetrahydrate (15.4 g, 0.038 mol) andtriethylamine (50 mL) are added and the reaction is heated to reflux.After 1.5 h, air is bubbled (i.e., at reflux) for 4 h into the darkgreen reaction solution with aid of a gas dispersion tube (flow rate=20cm³/min). At this point, the reaction mixture is carefully monitored byUV/Visible spectroscopy (i.e., a spectrum is taken every 0.5-1 h, ˜1drop diluted in 4-5 mL MeOH). The reaction is deemed complete by UV/Vis(In MeOH ratio: 342 nM/472 nm=0.22-0.24) after 4 h. The dark greenreaction is cooled to room temperature, filtered through Celite into a 2L round bottom flask, and the solvent removed under reduced pressure.The dark green solid is suspended in acetone (1 L) and the resultingslurry is stirred for 1 h at room temperature. The suspension isfiltered to remove the red/brown impurities (incomplete oxidationproducts), the solids rinsed with acetone (200 mL), and air dried. Thecrude complex (35 g) is dissolved in MeOH (600 mL), stirred vigorouslyfor 15 min, filtered through Celite, and transferred to a 2 L Erlenmeyerflask. An additional 300 mL of MeOH and 90 mL water are added to theflask, along with acetic acid washed LZY-54 zeolite (150 g). Thesuspension is agitated with an overhead mechanical stirrer forapproximately 3-4 h. The zeolite extraction is deemed complete with theabsence of free Gd(III). [To test for free gadolinium, the crude FormulaI is spotted heavily onto a reverse phase TLC plate (Whatman KC8F,1.5×10 cm) and the chromatogram developed using 10% acetic acid inmethanol. The green complex moved up the TLC plate close to the solventfront. Any free gadolinium metal will remain at the origin under theseconditions. After developing the chromatogram, the plate is dried andthe lower ¼ of the plate stained with an Arsenazo III solution inmethanol (4 mg Arsenazo III in 10 mL methanol). A very faint blue spot(indicative of free metal) is observed at the origin against a pinkbackground indicating very little free gadolinium metal.] The zeolite isremoved through a Whatman #3 filter paper and the collected solidsrinsed with MeOH (200 mL). The dark green filtrate is loaded onto acolumn of Amberlite IRA-904 anion exchange resin (30 cm length×2.5 cmdiameter) and eluted through the resin (ca. 10 mL/min flow rate) into a2 L round bottom flask with 300 mL 1-butanol. The resin is rinsed withan additional 100 mL of MeOH and the combined eluent evaporated todryness under reduced pressure. The green shiny solid Formula I is driedin vacuo for several hours at 40° C., to a well stirred ethanoicsolution (260 mL of Formula I at 55°-60° C., n-heptanes (ca. 600 mL) isadded dropwise (flow=4 mL/min) from a 1 L pressure-equalizing droppingfunnel. During the course of 1.5 h (300 mL addition) the green Formula Ibegan to crystallize out of the dark mixture. After complete addition,the green suspension is cooled and stirred for 1 h at room temperature.The suspension is filtered, the solids rinsed with acetone (250 mL), anddried in vacuo for 24 h to afford 26 g (63%), UV/vis: [(MeOH) λ_(max)nm]: 316, 350, 415, 473, 739; FAB MS, (M-20 Ac)⁺: m/e 1030; HRMS, (M−20Ac)⁺: m/e 1027.4036 (calcd. for C₄₈H₆₆ ¹⁵⁵GdN₅O₁₀, 1027.4016). Anal.calcd. for [C₅₂H₇₂GdN₅O₁₄] 0.5H₂O: C, 53.96; H, 6.36; N, 6.05, Gd,13.59. Found: C, 53.73; H, 6.26; N, 5.82; Gd, 13.92.

[0392] Synthesis of the Lutetium(III) Complex of Formula H: Themacrocyclic ligand Formula H is oxidatively metalated usinglutetium(III) acetate hydrate (9.75 g, 0.0230 mol) and triethylamine (22mL) in air-saturated methanol (1500 mL) at reflux. After completion ofthe reaction (as judged by the optical spectrum of the reactionmixture), the deep green solution is cooled to room temperature,filtered through a pad of celite, and the solvent removed under reducedpressure. The dark green solid is suspended in acetone (600 mL, stirredfor 30 min at room temperature, and then filtered to wash away thered/brown impurities (incomplete oxidation products and excesstriethylamine). The crude complex is dissolved into MeOH (300 mL,stirred for −30 min, and then filtered through celite into a 1 LErlenmeyer flask. An additional 50 mL of MeOH and 50 mL of water areadded to the flask along with acetic acid washed LZY-54 zeolite (40 g).The resulting mixture is agitated or shaken for 3 h, then filtered toremove the zeolite. The zeolite cake is rinsed with MeOH (100 mL and therinse solution added to the filtrate. The filtrate is first concentratedto 150 mL and then loaded onto a column (30 cm length×2.5 cm diameter)of pretreated Amberlite IRA-904 anion exchange resin (resin in theacetate form). The eluent containing the bis-acetate lutetium(III)texaphyrin complex is collected, concentrated to dryness under reducedpressure, and recrystallized from anhydrous methanol/t-butylmethyl etherto afford 11.7 g (63%) of a shiny green solid. For the complex: UV/vis:[(MeOH) λ_(max) nm (log ∈)]: 354,414, 474 (5.10), 672, 732; FAB MS,[IM-OAc⁻]⁺: m/e 1106.4; HRMS, (M-OAc⁻]⁺: m/e 1106.4330 (calcd. for[C₄₈H₆₆N₅;O₁₀Lu(OAc)]⁺, 1106.4351). Anal. calcd. for[C₄₈H₆₆N5O₁₀Lu](OAc)₂H2O; C, 52.74; H, 6.30; N, 5.91. Found: C, 52.74;H, 6.18; N, 5.84.

EXAMPLE 2 Synthesis of a T2B1 TXP Metal Complex-Oligonuleotide Conjugate

[0393] FIGS. 31A-H illustrate the synthesis of a light activated drugconjugate. The light activated drug is a texaphyrin coupled with anoligonucleotide which is complementary to a DNA site. As a result, thelight activated drug conjugate can bind the complementary DNA site andwill cleave the site upon activation by ultrasound. (Note: References to“Formula A,” “Formula B,” etc. relate to FIGS. 31A, 31B, etc.)

[0394] Synthesis of 4-Amino-1-[1-(ethyloxy)acetyl-2-oxy]-3-nitrobenzene(Formula B of FIG. 19), n=1. Potassium carbonate (14.0 g, 101 mmol) and4-amino-3-nitrophenol (Formula A) (10.0 g, 64.9 mmol) are suspended in150 mL dry acetonitrile. Ethyl-2-iodoacetate (10 mL, 84.5 mmol) (orethyl iodobutyrate maybe used, in that case n=3) is added via syringe,and the suspension is stirred at ambient temperature for ca. 21 h.Chloroform (ca. 375 mL) is added and is used to transfer the suspensionto a separatory funnel, whereupon it is washed with water (2xca. 100mL). The water washes are in turn washed with CHCl₃ (ca. 100 mL) and thecombined CHCl₃ extracts are washed with water (ca. 100 mL). Solvents areremoved on a rotary evaporator, and the residue is redissolved in CHCl₃(ca. 500 mL) and precipitated into hexanes (1.5 L). After standing twodays, the precipitate is filtered using a coarse fritted funnel anddried in vacuo to provide 14.67 g (Formula B), n=1 (94.1%). TLC:R_(f)=0.43, CHCl₃.

[0395] Synthesis of 4-Amino-1-[1-(hydroxy)acetyl-2-oxy]-3-nitrobenzene(Formula C), n=1. 4-Amino-1-[1-(ethyloxy)acetyl-2-oxy]-3-nitrobenzene(Formula B), n=1, (10.00 g, 37.3 mmol) is dissolved in tetrahydrofuran(100 mL), aqueous sodium hydroxide(1M solution, 50 mL) is added and thesolution is stirred at ambient temperature for ca. 21 h. Tetrahydrofuranis removed on a rotary evaporator, and water (100 mL) is added. Thesolution is washed with CHCl₃ (ca. 200 mL), the neutralized by additionof hydrochloric acid (1M solution, 50 mL). The precipitate which formedis filtered after standing a few minutes, washed with water, and driedin vacuo to provide 8.913 g compound (Formula C), n=1 (99.5%). TLC:R_(f)=0.65, 10% methanol/CHCl₃.

[0396] Synthesis of16-[1-(Hydroxy)acetyl-2-oxy]-9,24-bis(3-hydroxypropyl)-4,5-diethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1^(3,6).1^(8,11).0^(14,19)]heptacosa-3,5,8,10,12,14(19),15,17,20,22,24-undecaene (Formula E), n−1: 4-Amino-1-[1-(hydroxy)acetyl-2-oxy]-3-nitrobenzene (Formula C), n−1. (1,800 g, 8.49 mmol) isdissolved in methanol (100 mL) in a 1L flask. Palladium on carbon (10%,180 mg) is added, and the atmosphere inside the flask is replaced withhydrogen at ambient pressure. A grey precipitate is formed after ca. 3h, and the supernatant is clear. Methanol is removed in vacuo, takingprecautions to prevent exposure to oxygen, and the compound is driedovernight in vacuo. Isopropyl alcohol (500 mL) and HCl (12M, 400 μL) areadded, and the suspension is allowed to stir for ca. 15′.2,5-Bis[(3-hydroxypropyl-5-formyl-4-methylpyrrol-2-y)methyl]-3,4-diethylpyrrole(Formula D) (n=1) (4.084 g, 8.49 mmol) is added, and the reactionstirred at room temperature under argon for 3 hours. Hydrochloric acidis again added (12M, 400 μL) and the reaction again is allowed to stirfor an additional 3.5 h. The resulting red solution is filtered throughcelite, and the filtercake is washed with isopropyl alcohol until thefiltrate is colorless. Solvent is reduced to a volume of ca. 50 mL usinga rotary evaporator, whereupon the solution is precipitated into rapidlystirring Et₂O (ca. 700 mL). Formula E (n=1) is obtained as a red solid(5.550 g, 98.4%) upon filtering and drying in vacuo. TLC: R_(f)=0.69,20% methanol/CHCL₃ (streaks, turns green on plate with I₂).

[0397] Synthesis of metal complex of 16-[1-(hydroxy)acetyl-2-oxy]-9,24-bis(3-hydroxypropyl)-4,5-diethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1^(3,6).1^(8,11).0^(14,19)]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridecaene (Formula F), n=1: Approximately equal molar amountsof the protonated form of the macrocycle,16-[1-(hydroxy)acetyl-2-oxy]-9,24-bis(3-hydroxypropyl)-4,5-diethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.0.1.1^(3,6).1^(8,11).0^(14,19)]heptacosa-3,5,8,10,12,14(19),15,17,20,22,24-undecaenehydrochloride (Formula E), n=1, and a metal acetate pentahydrate arecombined with triethylamine in methanol, and are heated to reflux underair for 5.5 h. The reaction is cooled to room temperature, and stored at−20° C., overnight. Solvent is removed on a rotary evaporator, acetoneis added, and the suspension is stirred on a rotary evaporator for 2 h.The suspension is filtered and the precipitate dried briefly in vacuo,whereupon a solution is formed in methanol (ca. 250 mL) and water (25mL). The pH is adjusted to 4.0 using HCl (1M), HCl-washed zeolite LZY54is added (ca. 5 g) and the suspension is stirred on the rotaryevaporator for ca. 6 h. Amberlite™ IRA-900 ion exchange resin (NaFtreated, ca. 5 g) is added, and the suspension is stirred for anadditional hour. The suspension is filtered, the resin is washed withmethanol (ca. 100 mL), and the filtrate is adjusted to pH 4.0 using HCl(1M). Solvents are removed on a rotary evaporator, using ethanol (abs.)to remove traces of water. After drying in vacuo, the compound isdissolved in methanol (25 mL) and precipitated into rapidly stirringEt₂O (300 mL). Formula F, n=1, is obtained as a precipitate afterfiltering and drying in vacuo. An analytical sample is prepared bytreating 50 mg of Formula F, n=1, dissolved in methanol (25 mL) withacetic acid-washed zeolite, then acetic acid-washed Amberlite™ for ca. 1h. After reducing methanol to a minimum volume, the solution isprecipitated into rapidly stirring Et₂O (70 mL), filtered, and dried invacuo.

[0398] Postsynthetic modification of oligodeoxynucleotide-amine (FormulaG) with metal complex (Formula F), n=1: The metal complex of16-[1-(hydroxy)acetyl-2-oxy]-9,24-bis(3-hydroxypropyl)-4,5-diethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.1.1^(3,6).1^(8,11).0^(14,19)]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridacaene(Formula F), n=1, (about 30 mol) and N-hydroxysuccinimide (43 μmol) aredried together overnight in vacuo. The compounds are dissolved indimethylformamide (anhydrous, 500 μL) and dicyclohexylcarbodiimide (10mg, 48 μmol) is added. The resulting solution is stirred under argonwith protection from light for 8 h, whereupon a 110 μL aliquot is addedto a solution of oligodeoxynucleotide (Formula G) (87 μmol) in a volumeof 350 μL of 0.4M sodium bicarbonate buffer in a 1.6 mL Eppendorf tube.After vortexing briefly, the solution is allowed to stand for 23 h withlight protection. The suspension is filtered through 0.45 μm nylonmicrofilterfuge tubes, and the Eppendorf tube is washed with 250 μLsterile water. The combined filtrates are divided into two Eppendorftubes, and glycogen (20 mg/mL, 2 μL) and sodium acetate (3M, pH 5.4 30μL) are added to each tube. After vortexing, ethanol (absolute, 1 mL) isadded to each tube to precipitate the DNA. Ethanol is decanted followingcentrifugation, and the DNA is washed with an additional 1 mL aliquot ofethanol and allowed to air dry. The pellet is dissolved in 50% formamidegel loading buffer (20 μL), denatured at 90° C. for ca. 2′, and loadedon a 20% denaturing polyacrylamide gel. The band corresponding toconjugate (Formula H), n=1, is cut from the gel, crushed and soaked in1×TBE buffer (ca. 7 mL) for 1-2 days. The suspension is filtered throughnylon filters (0.45 μm) and desalted using a Sep-pak™ reverse phasecartridge. The conjugate is eluted from the cartridge using 40%acetonitrile, lyophilized overnight, and dissolved in 1 mM HEPES buffer,pH 7.0 (500 μL). The solution concentration is determined using UV/visspectroscopy.

EXAMPLE 3 Synthesis of Texaphyrin Metal Complexes with Amine-, Thiol- orHydroxy-Linked Oligonucleotides

[0399] Amides, ethers, and thioethers are representative of linkageswhich may be used for coupling site-directing molecules such asoligonucleotides to light activated drugs such as texaphyrin metalcomplexes as illustrated in FIGS. 32A-F. (Note: References to “FormulaA,” “Formula B,” etc. relate to FIGS. 32A, 32B, etc.). Oligonucleotidesor other site-directing molecules functionalized with amines at the5′-end, the 3′-end, or internally at sugar or base residues are modifiedpost-synthetically with an activated carboxylic ester derivative of thetexaphyrin complex. In the presence of a Lewis acid such as FeBr₃, abromide derivatized texaphyrin (for example, Formula C of FIG. 32) willreact with an hydroxyl group of an oligonucleotide to form an etherlinkage between the texaphyrin linker and the oligonucleotide.Alternatively, oligonucleotide analogues containing one or morethiophosphate or thiol groups are selectively alkylated at the sulfuratom(s) with an alkyl halide derivative of the texaphyrin complex.Oligodeoxynucleotide-complex conjugates are designed so as to provideoptimal catalytic interaction between the targeted DNA phosphodiesterbackbone and the texaphyrino.

[0400] Oligonucleotides are used to bind selectively compounds whichinclude the complementary nucleotide or oligo- or polynucleotidescontaining substantially complementary sequences. As used herein, asubstantially complementary sequence is one in which the nucleotidesgenerally base pair with the complementary nucleotide and in which thereare very few base pair mismatches. The oligonucleotide may be largeenough to bind probably at least 9 nucleotides of complementary nucleicacid.

[0401] For general reviews of synthesis of DNA, RNA, and theiranalogues, see Oligonucleotides and Analogues, F. Eckstein, Ed., 1991.IRL Press, New York; Oligonucleotide Synthesis, M. J. Gait, Ed., 1984,IRL Press Oxford, England; Caracciolo et al. (1989); BioconjugateChemistry, Goodchild, J. (1990); or for phosphonate synthesis,Matteucci, Md. et al., Nucleic Acids Res. 14:5399 (1986) (thesereferences are incorporated by reference herein).

[0402] In general, there are three commonly used solid phase-basedapproaches to the synthesis of oligonucleotides containing conventional5′-3′ linkages. These are the phosphoramidite method, the phosphonatemethod, and the triester method.

[0403] A brief description of a current method used commercially tosynthesize oligomeric DNA is as follows: Oligomers up to ca. 100residues in length are prepared on a commercial synthesizer, eg.,Applied Biosystems Inc. (ABI) model 392, that uses phosphoramiditechemistry. DNA is synthesized from the 3′ to the 5′ direction throughthe sequential addition of highly reactive phosphorous(III) reagentscalled phosphoramidites. The initial 3′ residue is covalently attachedto a controlled porosity silica solid support, which greatly facilitatesmanipulation of the polymer. After each residue is coupled to thegrowing polymer chain, the phosphorus(III) is oxidized to the morestable phosphorus(V) state by a short treatment with iodine solution.Unreacted residues are capped with acetic anhydride, the 5′-protectivegroup is removed with weak acid, and the cycle may be repeated to add afurther residue until the desired DNA polymer is synthesized. The fulllength polymer is released from the solid support, with concomitantremoval of remaining protective groups, by exposure to base. A commonprotocol uses saturated ethanolic ammonia.

[0404] The phosphonate based synthesis is conducted by the reaction of asuitably protected nucleotide containing a phosphonate moiety at aposition to be coupled with a solid phase-derivatized nucleotide chainhaving a free hydroxylphosphonate ester linkage, which is stable toacid. Thus, the oxidation to the phosphate or thiophosphate can beconducted at any point during synthesis of the oligonucleotide or aftersynthesis of the oligonucleotide is complete. The phosphonates can alsobe converted to phosphoramidate derivatives by reaction with a primaryor secondary amine in the presence of carbon tetrachloride.

[0405] In the triester synthesis, a protected phosphodiester nucleotideis condensed with the free hydroxyl of a growing nucleotide chainderivatized to a solid support in the presence of coupling agent. Thereaction yields a protected phosphate linkage which may be treated withan oximate solution to form unprotected oligonucleotide.

[0406] To indicate the three approaches generically, the incomingnucleotide is regarded as having an “activated” phosphite/phosphategroup. In addition to employing commonly used solid phase synthesistechniques, oligonucleotides may also be synthesized using solutionphase methods such as diester synthesis. The methods are workable, butin general, less efficient for oligonucleotides of any substantiallength.

[0407] Preferred oligonucleotides resistant to in vivo hydrolysis maycontain a phosphorothioate substitution at each base (J. Org. Chem.55:4693-469, (1990) and Agrawal, (1990)). Oligodeoxynucleotides or theirphosphorothioate analogues may be synthesized using an Applied Biosystem380B DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.).

EXAMPLE 4 Synthesis of Diformyl Monoacid Tripyrrane (FIG. 33. Formula H)and Oligonucleotide Conjugate (FIG. 33. Formula J)

[0408] The present example provides for the synthesis of a lightactivated drug conjugate. The light activated drug conjugate includes aoligonucleotide acting as a site directing molecule coupled with thetripyrrane portion of a texaphyrin as illustrated in FIG. 33A-J. (Note:References to “Formula A,” “Formula B,” etc. relate to FIGS. 33A, 33B,etc.).

[0409] Synthesis of Dimethylester Dibenzylester Dipyrromethane (FormulaB): A three-neck 1000 mL round-bottom flask set with a magnetic stirringbar, a thermometer, a heating mantle, and a reflux condenser attached toan argon line is charged with methylester acetoxypyrrole (Formula A)(100.00 g; 267.8 mmol), 200 proof ethyl alcohol (580 mL), and deionizedwater (30 mL.) The reaction mixture is heated up and when the resultingsolution begins to reflux, 12N aq. hydrochloric acid (22 mL) is addedall at once. The flask contents are stirred under reflux for two hours.The heating element is replaced by a 0° C. bath and the resulting thickmixture is stirred for two hours prior to placing it in the freezerovernight.

[0410] The mixture is filtered over medium fritted glass funnel, pressedwith a rubber dam, and washed with hexanes until the filtrate comes outcolorless. The collected solids are set for overnight high vacuum dryingat 30° C. to afford slightly yellowish solids (65.85 g, 214.3 mmol,80.0% yield.)

[0411] Synthesis of Dimethylester Diacid Dipyrromethane, Formula C: Allthe glassware is oven dried. A three-neck 2000 mL round-bottom flask setwith a magnetic stirring bar, a hydrogen line, and a vacuum line ischarged with dimethylester dibenzylester dipyrromethane (Formula B)(33.07 g, 53.80 mmol), anhydrous tetrahydrofuran (1500 mL), and 10%palladium on charcoal (3.15 g.) The flask is filled with dry hydrogengas after each of several purges of the flask atmosphere prior tostirring the reaction suspension under a hydrogen atmosphere for 24hours.

[0412] The solvent of the reaction suspension is removed under reducedpressure. The resulting solids are dried under high vacuum overnight.

[0413] The dry solids are suspended in a mixture of saturated aqueoussodium bicarbonate (1500 mL) and ethyl alcohol (200 mL), and stirred atits boiling point for five minutes. The hot suspension is filtered overcelite. The filtrate is cooled down to room temperature and acidified topH 6 with 12N aqueous hydrochloric acid. The resulting mixture isfiltered over medium fritted glass. The collected solids are dried underhigh vacuum to constant weight (21.63 g, 49.78 mmol, 92.5% yield.)

[0414] Synthesis of Methylester Dibenzylester Tripyrrane, Formula E: Athree-neck 2000 mL round-bottom flask set with a heating mantle, amagnetic stirring bar, a thermometer, and a reflux condenser attached toan argon line is charged with dimethyleser diacid dipyrromethane(Formula C) (21.00 g, 48.33 mmol), ethyl acetoxy pyrrole (Formula D)(30.50 g), p-toluenesulfonic acid monohydrate (1.94 g), trifluoroaceticacid (39 mL), and methyl alcohol (1350 mL.) The flask contents areheated and stirred under reflux for two hours. The heating element isreplaced with a 0° C. bath and the stirring is continued for half anhour prior to placing the resulting mixture in a freezer overnight.

[0415] The cold mixture is filtered over medium fritted glass. Thecollected solids are washed with hexanes and dried under high vacuumovernight (13.05 g, 19.25 mmol, 39.85 yield).

[0416] Synthesis of Methylester Diacid Tripyrrane, Formula F: All theglassware is oven dried. A three-neck 500 mL round-bottom flask set witha magnetic stirring bar, a hydrogen line, and a vacuum line is chargedwith methylester dibenzylester tripyrrane (Formula E) (12.97 g, 19.13mmol), anhydrous tetrahydrofuran (365 mL), and 10% palladium on charcoal(1.13 g.) The flask is filled with dry hydrogen gas after each ofseveral purges of the flask atmosphere prior to stirring the reactionsuspension for 24 hours under a hydrogen atmosphere at room temperature.

[0417] The reaction suspension is filtered over celite. The solvent ofthe filtrate is removed under reduced pressure to obtain a foam which isdried under high vacuum overnight (10.94 g, 21.99 mmol, 87.0% pure.)

[0418] Synthesis of Monoacid Tripyrrane, Formula H: All the glassware isoven dried. A three-neck 500 mL round-bottom flask set with a mechanicalstirrer, a thermometer, a 0° C. bath, and an additional funnel set withan argon line is charged with methylester diacid tripyrrane (Formula F)(10.20 g, 17.83 mmol). Trifluoroacetic acid (32.5 mL) is dripped intothe reaction flask from the addition funnel over a 45 minute periodkeeping the flask contents below 5° C. The resulting reaction solutionis stirred at 0° C. for 15 minutes, and then at 20° C. for three hours.Triethylorthoformate (32.5 mL) is dripped into the flask from theaddition funnel over a 20 minute period keeping the flask contents below−25° C. by means of a dry ice/ethylene glycol bath. The reactionsolution is stirred for one hour at −25° C. and then a 0° C. bath is setup. Deionized water (32.5 mL) is dripped into the reaction flask fromthe addition funnel keeping the flask contents below 10° C. Theresulting two phase mixture is stirred at room temperature for 75minutes and then added 1-butanol (200 mL.) The solvents are removedunder reduced pressure. The resulting dark oil is dried under highvacuum overnight to obtain black solids (11.64 g.)

[0419] A three-neck 2000 mL round-bottom flask set with a thermometer, aheating mantle, a magnetic stirring bar, and a reflux condenser attachedto an argon line, is charged with the crude methylester diformyltripyrrane (Formula G) (11.64 g), methyl alcohol (900 mL), deionizedwater (60 mL), and lithium hydroxide monohydrate (4.7 g.) The flaskcontents are heated, stirred under reflux for two hours, cooled down toroom temperature, added deionized water (250 mL), acidified with 12N aq.HCL to pH 5, and then stirred at 0° C. for one hour. The resultingmixture is filtered over medium fritted glass funnel. The collectedsolids are dried under high vacuum to constant weight prior to theirpurification by column chromatography (silica gel, MeOH in CH₂Cl₂,0-10%; 3.64 g, 8.06 mmol, 45.2% yield.)

[0420] The monoacid tripyrrane (Formula H) is condensed with aderivatized ortho-phenylene diamine to form a nonaromatic precursorwhich is then oxidized to an aromatic metal complex, for example,Formula I. An oligonucleotide amine may be reacted with the carboxylicacid derivatized texaphyrin Formula I to form the conjugate Formula Jhaving the site-directing molecule on the T (tripyrrane) portion of themolecule rather than the B (benzene) portion.

EXAMPLE 5

[0421] The following example describes the synthesis of an emulsionincluding tin ethyl etiopurpurin (SnEt₂) which is illustrated in FIG.34.

[0422] Several emulsions are prepared as described above. In 5 ml glasstubes, medium chain length oil known as MCT oil (Miglyol 801, H{umlautover (ν)}ls America, Piscataway, N.J.) is combined with 10 mg/gm SnEt₂plus excipients as described above. Certain emulsions also includedadditional excipients in the following concentrations: ethanol at mg/gmoil; egg phospholipids at 75 mg/gm oil; and sodium cholate at 10 mg/gmoil. After incubating for 30 minutes at 55° C., the tubes standovernight at room temperature (19°-22° C.). The tubes are centrifuged toremove bulk precipitates, and supernatants are filtered through 0.45 μmnylon membrane to remove any undissolved drug. Aliquots of filtrate arethen diluted in chloroform:isopropyl alcohol (1:1) forspectrophotometric determination of drug concentration (absorbance at662 nm). Reference standards are prepared with known concentrations ofSnEt₂ in the same solvent.

[0423] The concentration of SnEt₂ in each of the emulsions isillustrated in Table 2. As illustrated, the concentration of SnEt₂ inthe emulsion can be more than ten times the concentration in MCT oilalone. TABLE 2 Drug Solubility in Oil SnEt₂ SnEt₂ Excipient CombinationAdded to Concentration Concentration MCT Oil mg/gm oil Normalized MCToil alone 0.38 1.00 + ethanol 0.28 0.74 + egg phospholipids (EYP) 0.892.34 + Na cholate 1.17 3.08 + ethanol + EYP 1.37 3.61 + EYP + Na cholate1.77 4.66 + ethanol + Na cholate 2.20 5.79 + ethanol + EYP + Na cholate4.92 12.95

EXAMPLE 6

[0424] This example illustrates relative efficiencies of several bilesalts. Mixtures of MCT oil, egg phospholipids, ethanol, and SnEt₂ areincubated with different bile salts, all at 4.6 nM¹, under the sameconditions described above. As shown in Table 3, sodium cholate is themost efficient solubilizer. Cholic acid lacks solubilizing action in theoil. TABLE 3 Sodium Cholate is the Most Efficient Co-Solubilizer forSnEt₂ SnEt₂ SnEt₂ Concentration Concentration Bile compound mg/gm oilNormalized None 1.26 1.00 Na Tauracholate 1.13 0.90 Cholic acid 1.331.06 Na glycocholate 2.22 1.76 Na deoxycholate 2.31 1.83 Na cholate 3.702.94

EXAMPLE 7

[0425] The following Example illustrates the preparation of liposomesincluding BPD-MA (See FIG. 17) as a light activated drug. A 100-ml batchof BPD-MA liposomes is prepared at room temperature (about 20° C.) usingthe following general procedure. BPD-MA, butylated hydroxytoluene(“BHT”), ascorbyl palmirate, and the phospholipids DMPC and EPG aredissolved in methylene chloride. The molar ratio of light activateddrug: EPG:DMPC is 1.0:3.7 and has the compositions illustrated in Table4. TABLE 4 Light activated drug  0.21 g EPG  0.68 g DMPC  1.38 g BHT0.0002 g Ascorbic acid 6-palmitate  0.002 g Lactose NF crystallineinjectable    10 g Water for injection   100 ml

[0426] Using the above formulation, the total lipid concentration (%w/v) is about 2.06. The resulting solution is filtered through a 0.22 μmfilter and then dried under vacuum using a rotary evaporator. Drying iscontinued until the amount of methylene chloride in the solid residue isno longer detectable by gas chromatography.

[0427] A 10% lactose/water-for-injection solution is then prepared andfiltered through a 0.22 μm filter. Instead of being warmed to atemperature of about 35° C., the lactose/water solution is allowed toremain at room temperature (about 25° C.) for addition to the flaskcontaining the solid residue of the light activated drug/phospholipid.The solid residue is dispersed in the 10% lactose/water solution at roomtemperature, stirred for about one hour, and passed through aMicrofluidizer™ homogenizer three to four times with the outlettemperature controlled to about 200°-250° C. The solution is thenfiltered through a 0.22 μm Durapore, hydrophilic filter.

[0428] The filterability of the composition in g/cm² is typicallygreater than about 10. Moreover, the yield is about 100% by HPLCanalysis, with light activated drug potency typically being maintainedeven after sterile filtration. Average particle sizes vary from about150 to about 300 nm (±50 nm).

EXAMPLE 8

[0429] The following Example describes the delivery of a light activateddrug to an atheroma. An emulsion is prepared having about 0.6 g SnEt₂/mlof emulsion and about 20 g of MCT oil based hydrophobic phase/ml ofemulsion. The catheter illustrated in FIG. 7C is positioned in a vesselof the cardiovascular system using over the guidewire techniques. Thecatheter is positioned such that the media delivery port is adjacent tothe atheroma using radiopaque markers on the catheter and the balloon isexpanded into contact with the vessel wall. The emulsion is deliveredvia the third utility lumen 16B of the catheter 10. After the deliveryof the emulsion, the ultrasound energy is delivered at about 0.3 W/cm²at a frequency of approximately 1.3 MHz for about ten minutes. After thedelivery of ultrasound energy has concluded, the catheter is withdrawnfrom the vasculature of the tumor.

EXAMPLE 9

[0430] The following Example describes the delivery of a light activateddrug to a tumor. An emulsion is prepared having approximately 0.8 gSnEt₂/ml of emulsion and approximately 30 g of MCT oil based hydrophobicphase/ml of emulsion. The catheter 10 illustrated in FIG. 3A ispositioned in the vasculature of a tumor using over the guidewiretechniques. The catheter is positioned such that the media delivery portis within the tumor using radiopaque markers included on the catheter.The prepared emulsion is delivered into the vasculature of the tumor viathe utility lumen 16A. After the delivery of the emulsion, theultrasound energy is delivered at about 0.3 W/cm² at a frequency ofapproximately 1.3 MHz for about fifteen minutes. After the delivery ofultrasound energy has concluded, the catheter is withdrawn from thevascular system of the patient.

EXAMPLE 10

[0431] The following Example describes the delivery of a light activateddrug to a potential restenosis site. An emulsion is prepared havingapproximately 0.6 g SnEt₂/ml of emulsion and approximately 30 g of MCToil based hydrophobic phase/ml of emulsion. The catheter illustrated inFIG. 7C is positioned in the vasculature of a patient using over theguidewire techniques. The catheter is positioned such that the mediadelivery port is adjacent to a portion of the vessel which waspreviously treated with balloon angioplasty and the balloon is expandedinto contact with the vessel wall. The prepared emulsion is deliveredinto the vasculature of the patient via the third utility lumen 16B.Ultrasound energy is delivered from the ultrasound assembly to thepotential restenosis site at about 0.3 W/cm² at a frequency ofapproximately 1.3 MHz for about ten minutes. After the delivery ofultrasound energy has concluded, the catheter is withdrawn from thevascular system of the patient.

EXAMPLE 11

[0432] The following Example describes the delivery of a light activateddrug to an atheroma. Liposomes are prepared including BPD-MA (See FIG.17) as the light activated drug and DMPC and EPG as the phospholipids.The molar ratio of BPD-MA:EPG:DMPC is about 1:3:7. The catheterillustrated in FIG. 7C is positioned in a vessel of the cardiovascularsystem using over the guidewire techniques. The catheter is positionedsuch that the media delivery port is adjacent to the atheroma usingradiopaque markers included on the catheter and the balloon is expandedinto contact with the vessel. Ultrasound energy is delivered at about0.3 W/cm² at a frequency of approximately 1.3 MHz for about 20 minutesin order to rupture the liposomes and cause tissue death within theatheroma. After the delivery of ultrasound energy has concluded, thecatheter is withdrawn from the vascular system of the patient.

EXAMPLE 12

[0433] The following Example describes the delivery of a light activateddrug to a tumor. Liposomes are prepared including BPD-MA (See FIG. 17)as the light activated drug and DMPC and EPG as the phospholipids. Themolar ratio of BPD-MA:EPG:DMPC is about 1:3:7. The catheter illustratedin FIG. 8 is positioned in the vasculature of a tumor using over theguidewire techniques. The catheter is positioned such that the mediadelivery port is within the tumor using radiopaque markers included onthe catheter. Ultrasound energy is delivered at about 0.3 W/cm² at afrequency of approximately 1.3 MHz for about 20 minutes in order torupture the liposomes and cause tissue death within the atheroma. Afterthe delivery of ultrasound energy is concluded, the catheter iswithdrawn from the vasculature of the tumor.

EXAMPLE 13

[0434] The following Example describes the delivery of a light activateddrug to a potential restenosis site. Liposomes are prepared includingBPD-MA (See FIG. 17) as the light activated drug and DMPC and EPG as thephospholipids. The molar ratio of BPD-MA:EPG:DMPC is approximately1:3:7. The catheter illustrated in FIG. 7C is positioned in thevasculature of a patient using over the guidewire techniques. Thecatheter is positioned such that the media delivery port is adjacent toa portion of the vasculature which was previously treated with balloonangioplasty and the balloon is inflated into contact with the vesselwall. Ultrasound energy is delivered at about 0.3 W/cm² at a frequencyof approximately 1.3 MHz for about 15 minutes in order to rupture theliposomes and cause tissue death within the atheroma. After the deliveryof ultrasound energy is concluded, the catheter is withdrawn from thevasculature of the patient.

EXAMPLE 14

[0435] The following Example describes the delivery of a light activateddrug to an atheroma. Liposomes are prepared including BPD-MA (See FIG.17) as the light activated drug and DMPC and EPG as the phospholipids.The molar ratio of BPD-MA:EPG:DMPC is about 1:3:7. The phospholipids aresystemically delivered. The catheter illustrated in FIG. 7C ispositioned in the vasculature of a patient using over the guidewiretechniques. The catheter is positioned such that the media delivery portis adjacent to the atheroma and the balloon is inflated into contactwith the vessel wall. Ultrasound energy is delivered at about 0.3 W/cm²at a frequency of approximately 1.3 MHz for about 15 minutes. After thedelivery of ultrasound energy is concluded, the catheter is withdrawnfrom the vasculature of the patient.

EXAMPLE 14

[0436] The following Example describes the delivery of a light activateddrug to a tumor. Microbubbles are prepared including cisplatin andphotofrin according to the methods disclosed in U.S. Pat. No. 5,770,222.The microbubbles are systemically administered. The catheter illustratedin FIG. 1A is positioned within the vasculature of a tumor. Ultrasoundenergy is delivered at about 0.3 W/cm² at a frequency of approximately1.3 MHz for about 15 minutes. After the delivery of ultrasound energy isconcluded, the catheter is withdrawn from the vasculature of thepatient.

EXAMPLE 14

[0437] The following Example describes the delivery of a light activateddrug to a tumor. Microbubbles are prepared including cisplatin andphotofrin according to the methods disclosed in U.S. Pat. No. 5,770,222.The catheter illustrated in FIG. 3A is positioned within the vasculatureof a tumor. The microbubbles are delivered to the tumor via the secondutility lumen 16A of the catheter. Ultrasound energy is delivered atabout 0.3 W/cm² at a frequency of approximately 1.3 MHz for about 15minutes. After the delivery of ultrasound energy is concluded, thecatheter is withdrawn from the vasculature of the patient.

EXAMPLE 16

[0438] The following Example describes the delivery of a light activateddrug to a thrombosis. Microbubbles are prepared including heparin,photofrin and an albumin substrate. The microbubbles are systemicallyadministered. The catheter illustrated in FIG. 1A is positioned adjacentto the thrombosis. Ultrasound energy is delivered at about 0.2 W/cm² ata frequency of approximately 1.3 MHz for about 20 minutes. After thedelivery of ultrasound energy is concluded, the catheter is withdrawnfrom the vasculature of the patient.

EXAMPLE 17

[0439] Small hollow spheres covered with albumin were added to 5% humanserum albumin in beakers in the ratio of approximately 100,000,000spheres per 1 ml, and these were divided into one group that was treatedwith the ultrasound-sensitive substance Rose Bengal and one group thatwas untreated. The beakers containing the respective small hollowspheres were irradiated with 1 MHz ultrasound at 0.5 W/cm² for 30 secondand the number of small hollow spheres remaining after irradiation wascounted. While nearly all of the small hollow spheres coated with RoseBengal were ruptured, 70% of the untreated spheres kept their shape. Inthis manner, the presence of Rose Bengal was found to induce rupturingeffects not obtained with the mechanical energy of ultrasound. Note thatsimilar results were obtained using Eosin and other dyes instead of RoseBengal.

What is claimed is:
 1. A method of delivering a therapeutic compositionto a target site comprising: delivering the therapeutic compositioncomprising genetic material through a catheter to the target site; anddelivering ultrasound energy to the target site, wherein the catheterhas an elongated catheter body with at least one axial lumen fordelivery of genetic material therethrough, the catheter comprising atleast one ultrasound transducer coupled to an energy source, wherein theat least one ultrasound transducer generates a sufficient level ofultrasound energy.
 2. The method of claim 1, wherein the therapeuticcomposition further comprises a light activated compound.
 3. The methodof claim 1, wherein the genetic material is selected from the groupconsisting of DNA, RNA and analogs thereof.
 4. The method of claim 1,wherein the genetic material is synthetic.
 5. The method of claim 1,wherein the genetic material is recombinant.
 6. The method of claim 1,wherein the therapeutic composition further comprises a microbubble. 7.The method of claim 2, wherein the therapeutic composition furthercomprises a microbubble.
 8. The method of claim 1, wherein the geneticmaterial comprises an oligonucleotide.
 9. The method of claim 8, whereinthe oligonucleotide has an affinity for a DNA in the target site. 10.The method of claim 9, wherein the DNA is a viral DNA.
 11. The method ofclaim 9, wherein the DNA is an oncogene DNA.
 12. The method of claim 9,wherein the oligonucleotide is an antisense oligonucleotide.
 13. Themethod of claim 2, wherein the light activated drug is covalently boundto the genetic material.
 14. A therapeutic composition comprising alight activated drug in combination with a nucleic acid.
 15. The methodof claim 1, wherein the at least one ultrasound assembly is positionedabout a circumference of the elongated catheter body, the at least onesupport member supporting the at least one ultrasound transducer so asto define a chamber between the at least one transducer and the outercircumference of the elongated catheter body.
 16. The method of claim15, wherein the chamber is filled with a media that absorbs ultrasoundenergy such that a transmission of ultrasound energy from the ultrasoundtransducer to the elongated catheter body is reduced.
 17. The method ofclaim 16, wherein the media is a gas selected from the group consistingof helium, argon, air and nitrogen.
 18. The method of claim 16, whereinthe media is a solid media selected from the group consisting of siliconand rubber.
 19. The method of claim 15, wherein the chamber is evacuatedusing a negative pressure.
 20. The method of claim 1, wherein thecatheter further comprises: a balloon positioned about the circumferenceof the elongated catheter body; at least one media delivery port influid communication with the at least one axial lumen for delivery of anexpansion media to expand the balloon; and at least one media deliveryport in fluid communication with the at least one axial lumen fordelivery of a medicament.
 21. The method of claim 20, wherein theballoon is positioned about the ultrasound assembly.
 22. The method ofclaim 20, wherein the balloon is positioned about the circumference ofthe elongated catheter body adjacent to the ultrasound assembly.
 23. Themethod of claim 1, wherein the ultrasound transducer is configured todeliver ultrasound energy of approximately 0.3 W/cm² at a frequency ofapproximately 1.3 MHz.
 24. The method of claim 20, wherein pressure isused to drive the media across the balloon.
 25. The method of claim 6,wherein the microbubble comprises a lipid substrate.
 26. The method ofclaim 25, wherein the lipid substrate comprises a liposome.
 27. Themethod of claim 6, wherein the interior of the microbubble includes agas.